Biotechnological production of cyanophycin dipeptides

ABSTRACT

The present invention relates to a process for the enzymatic production of a dipeptide composition from a cyanophycin (CGP) or CGP-like polymer preparation by degrading the polymer preparation with an CGPase, a CGPase particularly adapted for said process, and the use of cyanophycin (CGP) or CGP-like polymers or fragments thereof, notably a dipeptide composition obtained by the process as defined above, as pharmaceutical composition, medicament, or as food or feed substitute.

The present invention relates to a process for the enzymatic productionof a dipeptide composition from a cyanophycin (CGP) or CGP-like polymerpreparation by degrading the polymer preparation with an CGPase, aCGPase particularly adapted for said process, and the use of cyanophycin(CGP) or CGP-like polymers or fragments thereof, notably a dipeptidecomposition obtained by the process as defined above, as pharmaceuticalcomposition, medicament, or as food or feed substitute.

BACKGROUND OF THE INVENTION

Three different poly(amino acid)s are known to occur naturally:poly(ε-L-lysine) (ε-PL), poly(γ-glutamic acid) (γ-PGA), and cyanophycin(CGP). Poly(amino acid)s are present in many environments and fulfildifferent functions for the producing organisms (Obst, M. et al.,Biomacromolecules 5:1166-1176 (2004)). For example, Cyanophycin(multi-L-arginyl-poly-[L-aspartic acid]), which is known also asCyanophycin Granule Polypeptide (CGP), which was discovered incyanobacteria more than 100 years ago (Borzi, A., Malpighia 1:28-74(1887)) provides the organism with nitrogen, carbon and energy. Itcontains five nitrogen atoms in every building block and therebyrepresents an ideal intracellular nitrogen reserve (Mackerras, A. H. etal., J. Gen. Microbiol. 136:2057-2065 (1990)). The biocompatibility andcomplete biodegradability of poly(amino acid)s make them idealcandidates for many applications in human life in the fields ofbiomedicine, agriculture, agrochemistry, personal care, and pharmacy(Obst, M. et al., Biomacromolecules 5:1166-1176 (2004)).

Several species of cyanobacteria including the blue green algaeSpirulina have been promoted as nutritional sources for humans andanimals (Kihlberg, R., A. Rev. Microbiol. 26:427-466 (1972)). CGP itselfwas discovered in 1887 in cyanobacteria. Most genera of cyanobacteriaharbor a functional cyanophycin synthetase gene (cphA) and synthesizeCGP (Mackerras, A. H. et al., J. Gen. Microbiol. 136:2057-2065 (1990)).Genes coding for CphA were identified also in heterotrophic bacteria(Krehenbrink, M. et al., Arch. Microbiol. 177:371-380 (2002); Fuser, G.et al., Macromol. Biosci. 7:278-296 (2007)). The branched polymer occursin the cytoplasm as insoluble intracellular membraneless granules(Allen, M. M. et al., J. Bacteriol. 154:1480-1484 (1983)). It consistsof equimolar amounts of arginine and aspartate arranged in the form ofpoly(aspartic acid) (PAA) backbone, with arginine moieties linked to theβ-carboxyl group of each aspartic acid by its α-amino group (Simon, R.D. et al., Biochim. Biophys. Acta 420:165-176 (1976)). For large scaleproduction, cyanobacterial cphA genes were heterologously cloned inEscherichia coli, Corynebacterium glutamicum, Ralstonia eutropha andPseudomonas putida. CGP from recombinant bacteria contains a littlelysine (Voss, I. et al., Metabol. Eng. 8:66-78 (2006)). CGP is widelyspread in different natural habitats and is degraded by intracellular orby extracellular CGPases (CphB, CphE, respectively). Bacteria possessingCphE were found in various habitats; CphE_(Pa) and CphE_(Bm) wereisolated and characterized from P. angulliseptica BI and B. megateriumBAC19, respectively (Obst, M. et al., J. Biol. Chem. 277:25096-25105(2002); Obst, M. et al., Biomacromolecules 5:153-161 (2004)). CGPdegradation occurs also in anaerobic habitats by strictly or facultativeanaerobic bacteria such as Sedimentibacter hongkongensis KI or P.alcaligenes DIP1, respectively (Obst, M. et al., Appl. Environ.Microbiol. 71:3642-3652 (2005); Sallam, A. et al., Submitted forpublication (2008)). All known CGPases produced water solubleβ-dipeptides from CGP which are then transported into the cells to befurther catabolized (Sallam, A. et al., Submitted for publication(2008)).

Protein digestion and transport is essential for life. In ruminants forinstance, the major part of dietary protein is degraded by the rumenflora to amino acids and peptides. Amino acids are incorporated intomicrobial protein or passed to next parts of the digestive tract orabsorbed directly across the rumen wall into the blood (Faix, {hacekover (S)}. et al., Acta Vet. Brno. 70:243-246 (2001)). However, Tri- anddipeptides are more efficiently utilized than free amino acids, havegreater nutritional value, are better absorbed [up to 185% greater thanfree amino acids (Adibi, S. A., J. Clin. Invest. 50:2266-2275 (1971))and retain more nitrogen than intact protein contributing to enhanceweight gain (Dock, D. B. et al., Biocell 28:143-150 (2004)). Absorptionstudies in patients with genetically impaired transport of certain aminoacids showed normal absorption of these amino acids if administered asdipeptides. This indicated the presence of specialized and effectivetransport systems for dipeptides (Adibi, S. A., Gastroenterology113:332-340 (1997)). Therefore, hydrolyzed protein diets are frequentlyapplied as feed additives to recovery malnourished cases (Dock, D. B. etal., Biocell 28:143-150 (2004)).

The semi-essential amino acid arginine plays several pivotal roles incellular physiology, and thus is applied in therapeutic regimens formany cardiovascular, genitourinary, gastrointestinal, or immunedisorders (for review see (Appleton, J., Altern. Med. Rev. 7:512-522(2002)). The essential amino acid lysine is known as food additive forhuman and animal, has antiviral activity against Herpes simplex virus,and improves calcium absorption in the small intestine, and hence actsagainst osteoporosis (Cynober, L. A., Metabolic and therapeutic aspectsof amino acids in clinical nutrition. 2nd ed. CRC Press LLC, Boca Raton,USA (2003)). The non-essential amino acid aspartate serves among othersas a precursor for L-arginine, for energy metabolism (Voet, D. et al.,Biochemistry. 3th ed. John Wiley and Sons Inc., New York (2004)), and isused in drug delivery for cations or for other amino acids (Cynober, L.A., Metabolic and therapeutic aspects of amino acids in clinicalnutrition. 2nd ed. CRC Press LLC, Boca Raton, USA (2003)). Because aminoacids have higher bioavailability in the dipeptide form, theiradministration as dipeptides was clinically approved and are availablein market products (Duruy, A. et al., Vie. Med. Int. 9:1589 (1965);Duruy, A., Med. Int. 1:203 (1966); Sellier, J., Rev. Med. Toulouse 5:879(1979); De-Aloysio, D. et al., Acta Eur. Fertil. 13:133-167 (1982);Rohdewald, P., Int. J. Clin. Pharmacol. Ther. 40:158-168 (2002); Lamm,S. et al., Eur. Bull. Drug Res. 11:29-37 (2003)).

Until now, no direct applications are known for CGP itself. Previousstudies on CGP were motivated by CGP as a potential source for abiodegradable PAA (Mooibroek, H. et al., Appl. Microbiol. Biotechnol.77:257-267 (2007)). The latter has many potential applications (Obst, M.et al., Biomacromolecules 5:1166-1176 (2004)) as a component in dialysismembranes, artificial skin and orthopedic implants or as drug carrier.PAA could also substitute non-biodegradable polyacrylates for which manytechnical applications are described. This is the first study on thebiodegradation of CGP by mammalian, avian and fish gut flora, andsubsequently on the potential applications of CGP and the dipeptidesthereof as nutritional and/or therapeutic additives.

Several samples of mammalian, avian and fish gut flora were investigatedfor cyanophycin degradation. All samples achieved complete anaerobic CGPdegradation over incubation periods of 12-48 h at 37° C. CGP degradingbacteria were found in all samples and were highly concentrated in cecumflora from rabbit and sheep and digestive tract flora from carp fish. Atotal of 62 axenic cultures were isolated and degraded CGP aerobically,46 thereof degraded CGP also anaerobically over incubation periodsranging from 24 h to 7 days. HPLC analysis revealed that all isolatesdegraded CGP to its constituting dipeptides. Eight strains wereidentified by 16S rDNA sequencing and were affiliated to the generaBacillus, Brevibacillus, Pseudomonas, Streptomyces and Micromonospora.CGP could be found in three different Spirulina platensis commercialproducts which contained 0.06-0.15% (wt/wt) CGP. It was now found thatCGP can be degraded extracellularly CGP degradation, as well as thefirst evidence on CGP biodegradability in the digestive tract, andsubsequently, the potential application of CGP and its dipeptides innutrition and therapy as highly bioavailable sources for arginine,lysine, aspartate and possibly other amino acids.

CGP accumulates in cyanobacteria during the transition from theexponential to the stationary growth phase (Mackerras, A. H. et al., J.Gen. Microbiol. 136:2057-2065 (1990); Sherman, D. M. et al., J. Phycol.36:932-941 (2000)). Most genera of cyanobacteria harbor a functionalcyanophycin synthetase gene (cphA) and synthesize CGP (Simon, R. D.1987. Inclusion bodies in the cyanobacteria: cyanophycin, polyphosphate,polyhedral bodies, pp. 199-225. In P. Fay and C. van Baalen (ed.), TheCyanobacteria, Elsevier, Amsterdam, The Netherlands; Allen, M. M. etal., Methods Enzymol. 167:207-213 (1988); Mackerras, A. H. et al., J.Gen. Microbiol. 136:2057-2065 (1990); Liotenberg, S. et al.,Microbiology 142:611-622 (1996); Wingard, L. L. et al., Appl. Environ.Microbiol. 68:1772-1777 (2002)). cphA genes were also identified inheterotrophic bacteria (Krehenbrink, M. et al., Arch. Microbiol.177:371-380 (2002); Ziegler, K. et al., Naturforsch. 57c:522-529(2002)). The polymer occurs in the cytoplasm as membraneless granulesand is insoluble at neutral pH as well as in physiological ionicstrength (Allen, M. M. et al., J. Bacteriol. 141:687-693 (1980)). CGPaccumulates under limiting conditions including low temperature, lowlight intensity, phosphorous or sulfur limitation (Stephan et al., Z.Naturforsch. 55:927-942 (2000)). In cyanobacteria, the molecular mass ofthe polymer strands range from 25 to 100 kDa (Simon, R. D., Biochim.Biophys. Acta 422:407-418 (1976)), while those from recombinant strainsexhibit a lower range (25 to 30 kDa) and polydispersity. Furthermore, itwas found that the polymer from recombinant strains contained lysine asan additional amino acid constituent (Ziegler, K. et al., Eur. J.Biochem. 254:154-159 (1998); Aboulmagd, E. et al., Biomacromolecules2:1338-1342 (2001)). CGP functions as a temporary nitrogen, energy andpossibly carbon reserve (Li, H. et al., Arch. Microbiol. 176:9-18(2001); Elbahloul, Y. et al., Appl. Environ. Microbiol. 71:7759-7767(2005)). Because CGP contains five nitrogen atoms in every buildingblock, it fulfills the criteria for the perfect intracellular nitrogenreserve (Simon, R. D. 1987. Inclusion bodies in the cyanobacteria:cyanophycin, polyphosphate, polyhedral bodies, pp. 199-225. In P. Fayand C. van Baalen (ed.), The Cyanobacteria, Elsevier, Amsterdam, TheNetherlands).

The intracellular degradation of CGP is catalyzed by highly specificcyanophycinases (CphB) occurring in the cytoplasm and proceeds via anα-cleavage mechanism resulting in the formation of β-dipeptides(Richter, R. et al., Eur. J. Biochem. 263:163-169 (1999)). CGPrepresents a valuable substrate also for bacteria not capable of CGPaccumulation (Obst, M. et al., Appl. Environ. Microbiol. 71:3642-3652(2005); Sallam, A., and A. Steinbüchel. 2007a). Clostridiumsulfatireducens sp. nov., a new mesophilic, proteolytic bacteriumisolated from a pond sediment, able to reduce thiosulfate, sulfur andtransiently sulfate), many of such bacteria were shown to possessextracellular cyanophycinases that degrades CGP to its utilizabledipeptides, which can be transported into the cell and further utilized(Sallam, A., and A. Steinbüchel. 2007b. Anaerobic and aerobicdegradation of cyanophycin by the denitrifying bacterium Pseudomonasalcaligenes strain DIP1-Role of other three co-isolates in the mixedbacterial consortium. Submitted for publishing.) Several examples ofthese enzymes were isolated and characterized, such as CphE_(Pa) fromthe Gram-negative bacterium Pseudomonas angulliseptica strain BI. Thisextracellular enzyme exhibited, similar to CphB, an α-cleavage mechanismfor CGP degradation (Obst, M. et al., J. Biol. Chem. 277:25096-25105(2002)).

Also Gram-positive bacteria were found to excrete CGPases when theextracellular CphE_(Bm) was isolated from Bacillus megaterium strainBAC19 (Obst, M. et al., Biomacromolecules 5:153-161 (2004)), bothCphE_(Pa) and CphE_(Bm) were identified as serine-type hydrolases.Recent studies revealed that extracellular CGP degradation can becatalyzed also by CGPases from strict as well as facultative anaerobicbacteria, such as Sedimentibacter hongkongensis strain KI (Obst, M. etal., Appl. Environ. Microbiol. 71:3642-3652 (2005)) and Pseudomonasalcaligenes strain DIP1 (Sallam, A., and A. Steinbüchel. 2007b.Anaerobic and aerobic degradation of cyanophycin by the denitrifyingbacterium Pseudomonas alcaligenes strain DIP1-Role of other threeco-isolates in the mixed bacterial consortium. Submitted forpublishing), respectively. All investigated CGPases yielded β-Asp-Argdipeptides as cleavage products, however, (Asp-Arg)₂ tetrapeptides wereadditionally detected in case of CphE_(Bm) (Obst, M. et al.,Biomacromolecules 5:153-161 (2004)).

Until recently, no practical applications were known for CGP itself orfor the dipeptides thereof. On contrast, economically importantapplications have been established for poly(aspartic acid) (PAA), whichis a structural element (polymer backbone) of CGP, as a substitute fornon-biodegradable polyacrylates (Schwamborn, M., Polym. Degrad. Stab.59:39-45 (1998)). PAA can be also employed in many fields includingpaper, paint and oil industries (reviewed by Joentgen, W. et al. 2003.Polyaspartic acids. pp. 175-499. In: S. R. Fahnestock and A. Steinbüchel(ed.), Biopolymers, vol 7. Wiley, Weinheim). Biomedical applicationshave also been described for PAA (Leopold, C. S. et al., J.Pharmacokinet. Biopharm. 4:397-406 (1995); Yokoyama, M. et al., CancerRes. 6:1693-1700 (1990)). Only recently, biomedical applications forCGP-dipeptides and possibly for CGP itself were revealed, theseapplications depend in first place on the astonishing wide spread ofCGP-degrading bacteria in numerous investigated mammalian, avian, andfish flora, this indicated that CGP is probably degradable within therespective digestive tracts, On the other hand, the elevatedbioavailability of amino acids if administrated in the dipeptide ortripeptide form is a well known theory and is effectively applied inseveral therapeutic fields. Thus, CGP and/or its β-dipeptides can beconsidered as potential natural food and/or therapeutic additives forthe near future (Sallam, A., and A. Steinbüchel. 2007c. Potential ofcyanophycin and its β-dipeptides as possible additives in therapy, foodand feed industries).

The production and efficient isolation of CGP in semi-technical amountswas established only during the last few years. Several bacterialstrains of E. coli, Raistonia eutropha, Pseudomonas putida andAcinetobacter baylyi strain ADP1 were applied, the later showed themaximum CGP yield of about 46% (wt/wt) (Obst, M. et al., pp. 167-194. InJ. M. Shively (ed.), Inclusions in Prokaryotes, vol. 1. Springer-Verlag,Berlin, Heidelberg (2006)). However, the required substrates andcultivation conditions are also crucial factors for choosing theeconomically appropriate CGP-producer.

It was now found that pure CGP-dipeptides can be prepared in aneconomical large scale process which starts from CGP-containing biomassand ends with pure CGP-dipeptides. Because strain P. alcaligenes DIP1could show high enzyme productivity on simple growth requirements. Thisstrain was found ideal for such a technical process.

Cyanophycin contains five nitrogen atoms in every building block andtherefore accomplishes exactly the criteria for a perfect dynamicintracellular nitrogen reserve (Simon, R. D. 1987. Inclusion bodies inthe cyanobacteria: cyanophycin, polyphosphate, polyhedral bodies, pp.199-225. In P. Fay and C. van Baalen (ed.), The Cyanobacteria, Elsevier,Amsterdam, The Netherlands); its amount fluctuates according to theneeds of the cells (Carr, N. G. 1988. Nitrogen reserves and dynamicreservoirs in cyanobacteria, p. 13-21. In L. J. Rogers and J. R. Gallon(ed.), Biochemistry of the algae and cyanobacteria, Annual Proceedingsof the Phytochemical Society of Europe, Clarendon, Oxford.). The polymeraccumulates in cyanobacteria when the protein synthesis is diminishedeither naturally during the transition from the exponential to thestationary growth phase (Simon, R. D., Arch. Microbiol. 92:115-122(1973a)) or by addition of inhibitors of protein biosynthesis (e.g.chloramphenicol) (Ingram, L. O. et al., Arch. Microbiol. 81:1-12 (1972);Simon, R. D., J. Bacteriol. 114:1213-1216 (1973b)) and the polymerdisappears when balanced growth resumes (Mackerras, A. H. et al., J.Gen. Microbiol. 136:2057-2065 (1990)). CGP accumulation is also promotedby phosphorous limitation (Stephan et al., Z. Naturforsch. 55:927-942(2000)), sulfure limitation (Ariño, X. et al., Arch. Microbiol.163:447-453 (1995)), low temperature, low light intensity or acombination of these factors (Obst, M. et al., Biomacromolecules5:1166-1176 (2004)).

Different methods were developed for determination and quantification ofeither purified CGP or its content in cells. Arginine content of CGP wasquantified colorimetrically either in hydrolyzed or in unhydrolyzedpolymer by the Sakagushi reagent (Simon, R. D., J. Bacteriol.114:1213-1216 (1973b)). The amino acid constituents of the purifiedcyanophycin could be determined by HPLC (Aboulmagd, E. et al., Arch.Microbiol. 174:297-306 (2000)). For rapid and sensitive determination ofcyanophycin, a method based on ¹H nuclear magnetic resonance (NMR) wasdeveloped (Erickson, N. A. et al., Biochim. Biophys. Acta. 1536:5-9(2001)).

CGP degradation (intra- or extracellular) leads mainly to the release ofits utilizable dipeptides, these are then split intracellularly to theirconstituting amino acids to be engaged into cell metabolism.Intracellular degradation of cyanophycin is catalyzed by cyanophycinases(CphB). The first cyanophycinase was described in heterocysts andvegetative cells of Anabaena cylindrica by Gupta, M. et al., J. Gen.Microbiol. 125:17-23 (1981). The enzyme is a monomeric 29.4 kDa,serine-type, and a cyanophycin-specific exopeptidase, its maindegradation product was aspartate-arginine dipeptides via an α-cleavagemechanism (Richter, R. et al., Eur. J. Biochem. 263:163-169 (1999)). Inthe last few years, aerobic and anaerobic bacteria able to degradecyanophycin by extracellular cyanophycinases (CphE) were isolated (Obst,M. et al., J. Biol. Chem. 277:25096-25105 (2002)); Obst, M. et al.,Biomacromolecules 5:153-161 (2004); Obst, M. et al., Appl. Environ.Microbiol. 71:3642-3652 (2005); Sallam, A., and A. Steinbüchel. 2007b.Anaerobic and aerobic degradation of cyanophycin by the denitrifyingbacterium Pseudomonas alcaligenes strain DIP1-Role of other threeco-isolates in the mixed bacterial consortium. Submitted forpublishing). Similar to CphB, the previously characterized extracellularCGPases; CphE_(Pa) and CphE_(Bm), from Pseudomonas anguillisepticastrain B1 and Bacillus megaterium strain BAC19, respectively, wereidentified as serine-type, cyanophycin-specific enzymes and producedCGP-dipeptides as degradation products, however, (Asp-Arg)₂tetrapeptides were additionally detected in case of CphE_(Bm). Labelingstudies of CphE_(Pa) showed that the enzyme hydrolyses CGP at thecarboxyl-terminus and successively releases β-Asp-Arg dipeptides fromthe degraded polymer chain end (for review see Obst, M. et al.,Biomacromolecules 5:1166-1176 (2004)). Moreover, a third extracellularcyanophycinase (CphE_(al)) from Pseudomonas alcaligenes DIP1 wasrecently applied in crude form for the technical production ofCGP-dipeptides (Sallam, A., and A. Steinbüchel. 2008b. Biotechnologicalprocess for the technical production of β-dipeptides from cyanophycin.Under preparation).

The production and efficient isolation of CGP in semi-technical amountswere established only during the last few years. Several bacterialstrains of Escherichia coli, Ralstonia eutropha, Pseudomonas putida andAcinetobacter baylyi were successfully applied (Obst, M. et al.,Biomacromolecules 5:1166-1176 (2004)). However, the biotechnologicalrelevance of CGP was based theoretically on being a source forpoly(aspartic acid) which has high potential for industrial applications[e.g. for water treatment; paper and leather industries, as dispersingagent (Roweton, S. et al., J. Environ. Polym. Degrad. 5:175-181 (1997);Mooibroek, H. et al., Appl. Microbiol. Biotechnol. 77:257-267 (2007)) oras biodegradable substitute for polyacrylate (Schwamborn, M., Polym.Degrad. Stab. 59:39-45 (1998)). PAA has also potential biomedicalapplications as a component in dialysis membranes, artificial skin,orthopaedic implants and as drug carrier (Leopold, C. S. et al., J.Pharmacokinet. Biopharm. 4:397-406 (1995)).

As set forth above, biomedical applications for CGP-dipeptides andpossibly for CGP itself were revealed, this indicated that CGP isprobably degradable within the mammalian and fish digestive tracts; thisrepresented the polymer and the dipeptides thereof as potential naturalfood and/or therapeutic additives for the near future. Accordingly, alarge scale process for the production of dipeptides from CGP usingcrude CphE_(al) from P. alcaligenes strain DIP1 was recentlyconstructed. This original process comprised three phases; Phase I:large scale extraction and purification of CGP, Phase II: large scaleproduction of crude CphE_(al) powder, Phase III: degradation of CGP toits dipeptides was set up as described hereinbefore. It was now found,the latter two phases of the original process can be greatly optimizedfor future applications. Moreover, CphE_(al) was technically purifiedfrom the crude powder and the biochemical characteristics thereof wererevealed.

SUMMARY OF THE INVENTION

The present invention thus provides

(1) a process for the enzymatic production of a dipeptide compositionfrom a cyanophycin (CGP) or CGP-like polymer preparation, which processcomprises degrading the polymer preparation with an CGPase;(2) a preferred embodiment of the process of (1) above, wherein theCGPase(i) has a molecular weight of 45 kDa, an optimum temperature of 50° C.,and an optimum pH range of 7-8.5 and degrades CGP into β-Asp-Arg; and/or(ii) is the P. alcaligenes DIP1 CGPase CphE_(al) having been depositedwith the DSMZ as DSM 21533, or is a mutant, derivative or fragmentthereof capable of cleavage of CGp or CGP-like polymers into dipeptides;(3) a CGPase as defined in (2) above; and(4) a composition, pharmaceutical composition, medicament, food or feedsupplement comprising a cyanophycin (CGP) or a CGP-like polymer orfragments thereof;(5) the use of a cyanophycin (CGP) or a CGP-like polymer or fragmentsthereof for preparing a medicament for nutritional therapy, or as foodor feed supplement;(6) a method for nutritional therapy of a patient in need thereof, saidmethod comprising administering to the patient a suitable amount of acomposition comprising cyanophycin (CGP) or a CGP-like polymer orfragments thereof;(7) preferred embodiments of (4) to (6) above, wherein the composition,pharmaceutical composition, medicament, food or feed supplementcomprises dipeptides or a dipeptide mixture derived from the CGP or aCGP-like polymer by enzymatic proteolysis, preferably the dipeptidemixture being composed of β-aspartate-arginine and β-aspartate-lysineand/or being obtainable by the process of (1) or (2) above.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: Neighbour-joining tree based on 16S rDNA sequences showing theestimated phylogenetic relationships among the CGP degrading bacteriaisolated previously as well as during this study. Bolded strains wereisolated during this study. Underlined strains were previouslyinvestigated for CGP degradation (Obst, M. et al., J. Biol. Chem.277:25096-25105 (2002); Obst, M. et al., Biomacromolecules 5:153-161(2004); Obst, M. et al., Appl. Environ. Microbiol. 71:3642-3652 (2005);Sallam, A. et al., Submitted for publication (2008)). E. coli K12 wasused as outgroup. Accession numbers are given in parentheses. Bootstrapvalues are shown as percentages of 100 replicates. Bar, 2% sequencedivergence.

FIG. 2: Degradation halos caused by the extracellular CGPase form P.alcaligenes strain DIP1 on CGP-overlay agar plate; CphE: the crudepowder before the degradation phase (phase III), CphE R: the recoveredpowder after the degradation phase.

FIG. 3: Growth of P. alcaligenes strain DIP1 on different substrates.Cultivations were applied in 100 ml Klett-flasks with baffles; eachflask contained 10 ml of SM medium and 1 g l⁻¹ of the tested substrate.Experiments were performed in duplicates which were inoculated from apreculture grown under the same test conditions. Growth was monitored bythe increase in OD_(578 nm) after an incubation period of 24 h at 30° C.

FIG. 4: The required incubation periods for the complete degradation ofdifferent CGP concentrations (10-50 g/l) under the catalytic effect ofdifferent crude CphE concentrations (1-10 g/l). The reaction tubes wereincubated at 30° C. in a tube rotator with rotation rate of 3 rpm. Thehighest tested concentration of CGP (50 g/l) could be degraded within 10h in the presence of 2 g/l crude CphE powder.

FIG. 5: Batch fermentation of P. alcaligenes DIP1 in Biostat D650stirred tank reactor containing 420 l SM medium with g/l sodium citrateand inoculated with 4% (vol/vol) preculture. The preculture wascultivated in 2 l baffled-flasks containing 1 l of the same medium andincubated for 12 h at 30° C. The fermentation parameters and cultivationconditions in the Biostat D650 reactor were; pH of 6.9-7.5, temperatureof 30° C., and aeration at 0.2 vvm. pO₂ was set to a minimum of 40% andwas adjusted automatically by stirring which otherwise was kept at 100rpm. The arrow indicates the time of induction. * Optical density at 600nm (OD₆₀₀), ♦). * pH, ⊥). * Stirrer speed (rpm), ▴). * pO₂ (% ofsaturation), -). * Air flow (l min⁻¹), ).

FIG. 6: A continuous system for harvesting, concentration and desaltingof proteins in the fermentation supernatant of P. alcaligenes strainDIP1 (phase II). For harvesting, a CEPA Z41 continuous centrifuge wasused to separate the cells, the supernatant was collected in a central100 l tank. For concentration, a cross flow unit with a 30 kDa cassettewas connected to the central tank; the concentrated retentate wasre-pumped into the tank while the permeate was directly discarded. Theflow rate of the cross flow was adjusted to maintain only 50 l in thetank. The final concentrated 5 l were desalted with 5 bed volumes ofH₂O, frozen at −30° C., and lyophilized.

FIG. 7: TLC plates for the quality control of the producedCGP-dipeptides, I: Standard amino acids and direct dipeptide samplestaken after the tested filtration systems; 1: 30 kDa.-COP. cross flowcassette. 2: filter-membrane (10 kDa.-COP). 3: filter-membrane (5kDa.-COP). 4: filter-membrane (1 kDa.-COP). 5: filter-membrane (0.5kDa.-COP). 6: CGP-dipeptides (final charge after cross flow andlyophilization). II: Standard amino acids and hydrolyzed samples of; a:CGP-dipeptides (final charge after cross flow and lyophilization). b:Asp-Arg dipeptides. c: Asp-Lys dipeptides. (b, c; Sigma Aldrich,Deisenhofen, Deutschland). only one spot was indicated for all directsamples (I) while hydrolyzed samples showed typical spots for thestandard amino acids aspartate, arginine and lysine (II).

FIG. 8: Batch fermentation of E. coli DH1 (pMa/c5-914::cphA_(PCC6803))in Biostat D650 stirred tank reactor containing 400 l of 7% (vol/vol)protamylase medium with 100 mg l⁻¹ ampicillin. The preculture (4%,vol/vol) was prepared in 2 l flasks each containing 1 l of the samemedium as that for fermentation and incubated for 20 h at 30° C.Fermentation parameters and cultivation conditions in the Biostat D650reactor were pH 7.5, aeration at 0.17 vvm, pO₂ was kept constant at 20%and was adjusted automatically by stirring. Fermentation was run for 15h, the first 6 h at 30° C. then at 37° C. to induce expression ofCGP-synthetase. Turbidity (OD₈₅₀) (♦), pO₂ (% of saturation) (▾),Aeration (l/min) (▴), Stirring (rpm) (), Temperature (° C.) (▪).

FIG. 9: Phase-contrast micrograph of cells of E. coli DH1(pMA/c5.914::cphA_(PCC6803)) at the 15^(th) h of fermentation in BiostatD650 reactor on 7% (vol/vol) protamylasse medium. CGP grana appear aslight-reflecting accumulations in the cells. Bar; 10 μm.

FIG. 10: SDS-PAGE of purification steps of CphE_(al) from P. alcaligenesstrain DIP1 via specific binding on CGP. Gel A: SDS-PAGE stained bysilver nitrate method; M: molecular mass standard proteins, C: controlof crude CphE_(al), S1: supernatant sample immediately after mixing CGPand crude CphE_(al) together, S2: supernatant sample after 6 min bindingtime, S2″: same as S1 after 10 folds concentration, W1, W2: supernatantsamples after both washing steps. Gel B: SDS-PAGE with triple-volume ofthe purified CphE_(al) as in A and stained longer with silver nitrate.Few other low-concentrated protein bands can be observed in addition tothat of CphE_(al) at 45 kDa.

DETAILED DESCRIPTION OF THE INVENTION

In the process of aspect (1) of the invention the dipeptide compositionmay be composed of a single dipeptide or of a mixture of dipeptides. Itis however preferred that the dipeptides comprise amino acid residuesselected from aspartate, arginine, lysine and other amino acid residuespresent in the CGP-like polymer. Particularly preferred is that thedipeptides are selected from β-aspartate-arginine andβ-aspartate-lysine.

A “CGP” and “CGP-like polymer” according to the invention is a peptidicstructures essentially comprised of one or more dipeptide units,preferably said dipeptides units are composed of two of the followingamino acid residues aspartic acid, arginine, lysine, glutamic acid,citrulline, ornithine, canevanine and the like.

A great variety of CGPases known in the art may be utilized for the CGPdegradation (see Tables 2 and 4). It is, however, preferred that theCGPase is a CGPase from P. alcaligenes, particularly preferred from P.alcaligenes strain DIP1. According to aspect (2) of the invention theCGPase (i) has a molecular weight of 45 kDa, an optimum temperature of50° C., and an optimum pH range of 7-8.5 and degrades CGP intoβ-Asp-Arg; and/or (ii) is the P. alcaligenes DIP1 CGPase CphE_(al)having been deposited with the DSMZ as DSM 21533, or is a mutant,derivative or fragment thereof capable of cleavage of CGP or CGP-likepolymers into dipeptides. The mutants, derivatives or fragments of theaforementioned native CGPase include fragments (having at least 50consecutive amino acid residues of the native sequence, preferably N-and/or C-terminal truncation products, wherein up to 50, up to 30, or upto 10 terminal amino acid residues are removed), derivatives (notablyfusion products with functional proteins and peptides such as secretionpeptides, leader sequences etc., and reaction products with chemicalmoieties such as PEG, alcohols, amines etc.) and mutants (notablyaddition, substitution, inversion and deletion mutants, having at least80%, preferably at least 90%, most preferably at least 95% sequenceidentity with the native enzyme on the amino acid basis or wherein 1 to20, preferably 1 to 10, consecutive or separated amino acid residues areadded, substituted, inverted and/or deleted; for substitution mutantsconservative substitution is particularly preferred), provided, however,that said modified CGPases have the enzymatic activity of the nativeCGPase.

The process of aspects (1) and (2) of the invention may further comprisepreparing the CGP or CGP-like polymer preparation by culturing aprokaryotic or eukaryotic producing cell line. The producing cell linemay be any cell line capable of producing the CGP or CGP-like polymer.It is preferred that the producing cell line is selected fromEscherichia coli, Raistonia eutropha, Acinetobacter baylyi,Corynebacterium glutamicum, Pseudomonas putida, yeast strains, and plantbiomass. Particularly preferred producing cell lines are Raistoniaeutropha H16-PHB⁻4-Δeda (pBBR1MCS-2::cphA₆₃₀₈/edaH16) and E. coli DH1(pMa/c5-914::cphA_(PCC6803)).

The above process may further comprise the steps of isolating, purifyingand/or chemically modifying the CGP product obtained by cultivating theproducing cell line. Such isolation, purification and chemicalmodification separation may be effected by methods well established inthe art.

It is however preferred for the process of aspects (1) and (2) that theCGP product obtained by cultivating the producing cell line is directly,i.e. without isolation or purification subjected to degradation with theCGPase.

In another preferred embodiment the process of aspects (1) and (2)further comprises purifying or separating the degradation product and/orchemically modifying the degradation product. Again, such purification,separation or chemical modification may be effected by methods wellestablished in the art.

Aspect (3) of the invention pertains to a CGPase that

(i) has a molecular weight of 45 kDa, an optimum temperature of 50° C.,and an optimum pH range of 7-8.5 and degrades CGP into β-Asp-Arg; and/or(ii) is the P. alcaligenes DIP1 CGPase CphE_(al) having been depositedwith the DSMZ as DSM 21533, or is a mutant, derivative or fragmentthereof capable of cleavage of CGp or CGP-like polymers into dipeptides.As to the mutants, derivatives and fragments it is referred to thedefinition given above.

The pharmaceutical composition, medicament, food or feed supplementaccording to aspects (4), (5) and (6) of the invention may furthercontain pharmaceutically or dietetically acceptable suitable carriers,binders etc. They may further contain additional active compounds forthe respective pharmaceutical purpose.

The pharmaceutical composition is particularly suitably for nutritionaltherapy. The type of nutritional therapy of course depends on the aminoacids present within the Composition/medicament as will be apparent fromthe following:

Recent advances in nutritional therapy of critically ill patientsrendered a good understanding of the necessity of certain amino acidsfor maintaining tissue protein homeostasis during illness (Witte, M. B.and Barbul A., Wound Rep. Reg. 11:419-423 (2003)). Previously, aminoacids were either classified as nonessential (dispensable) or essential(non-dispensable). However, with better understanding of the in vivophysiology involving amino acids, an alternative classification wasproposed that redefines the requirements of certain amino acids as beingconditionally non-dispensable (Laidlaw, S. A. and Kopple, J. D., Am. J.Clin. Nutr. 46:593-605 (1987)). This made the use of such amino acids,solely or as part of a complete nutritional regimen, attractive toimprove nutritional outcome, immune response, and tissue recovery. Inthe following section, findings regarding the physiology and themechanism of action of the three amino acids, that also constitute CGP,are discussed. These amino acids are: the nonessential L-Aspartate, thesemi-essential L-Arginine, and the essential amino acid L-Lysine. Aspecial emphasis is given on arginine due to its numerous physiologicaleffects.

L-Aspartate: The nonessential L-Aspartate has a molecular mass of 133.10g/mol and is a dicarboxylic amino acid. Most L-Aspartate can be found inproteins while small amounts thereof are also found in the free form inbody fluids and in plants (Barrett, G. C. and Elmore D. T. Amino Acidsand Peptides. Cambridge University Press, Cambridge, UK (1998)).L-Aspartate is a constituent of the natural biopolymer CGP and thesynthetic sweetener Aspartame. Aspartic acid is slightly soluble inwater and more water-soluble in the salt form. Dietary aspartate isabsorbed from the small intestine by active transportation to enter theportal circulation then transported to the liver, where much of it ismetabolized to protein, purines, pyrimidines plants (Barrett, G. C. andElmore D. T. Amino Acids and Peptides. Cambridge University Press,Cambridge, UK (1998)). L-Aspartate can also serve as a source for energyin the citric acid cycle and thus is supposed to be effective againstfatigue (see also below: Asp-Arg). Aspartate is used in drug deliveryfor cations like Mg²⁺, K⁺, Ca²⁺, Zn²⁺, or for other amino acids toincrease their bioavailability (Cynober, L. A. Metabolic and therapeuticaspects of amino acids in clinical nutrition. 2nd ed. CRC Press LLC,Boca Raton, USA (2003)).

L-Arginine: L-Arginine is a strongly basic amino acid with a molecularmass of 174.2 g/mol and is found in most proteins. It contains fournitrogen atoms per molecule, and is therefore the most abundant nitrogencarrier in humans and animals (Appleton, J., Altern. Med. Rev. 7:512-522(2002)). Arginine is essential for fish (Ahmed, I. and Khan, M. A.,Aquacult. Nutr. 10:217-225 (2004)) whereas in mammals, it is consideredsemi-essential because it can be compensated via nutritional intake orvia de novo synthesis (endogenous). In the kidney, most endogenousarginine is derived from Citrulline, a by-product of glutaminemetabolism in the gut or liver. However, because arginine biosynthesisdoes not increase to compensate depletion or inadequate supply, dietaryintake (approximately 5-6 g/day for an average human, Witte, M. B. andBarbul. A., Wound Rep. Reg. 11:419-423 (2003)) remains the primarydeterminant of plasma arginine levels. About 50% of the ingestedarginine is directly utilized in the small bowel while the rest isreleased into the portal circulation. In general, about the half ofingested arginine is rapidly converted to ornithine, primarily by theenzyme arginase (Modolell, M. et al., Eur. J. Immunol. 25:1101-1104(1995); Witte, M. B. and Barbul. A., Wound Rep. Reg. 11:419-423 (2003)).Ornithine, in turn, can be metabolized to glutamate and proline, orthrough the enzyme ornithine decarboxylase into polyamines (Boutard, V.et al., J. Immunol. 155:2077-2084 (1995)). Rest arginine is processed byone of four other enzymes: nitric oxide synthase (to become nitricoxide), arginine:glycine amidinotransferase (to become creatine),arginine decarboxylase (to become agmatine), or arginyl-tRNA synthetase(to become arginyl-tRNA, a precursor to protein synthesis) (Vodovotz, Y.et al., J. Exp. Med. 178:605-613 (1993)).

Arginine has significant effects on endocrine functions in humans andanimals, particularly on adrenal and pituitary secretory functions.However, little is known about the exact mechanism by which arginineexerts these effects. Arginine is the biologic precursor of nitric oxide(NO), an endogenous messenger molecule involved in a variety ofendothelium-dependent physiological effects in the cardiovascular system(Witte, M. B. and Barbul. A., Wound Rep. Reg. 11:419-423 (2003)). Thus,many of the clinical effects of arginine are thought to be mediated byits effects on endothelial-derived relaxing factor. NO-synthase has twovariants (Rohdewald, P. and Ferrari V., Patent Application US2004137081(2004); the constitutive (cNOS) with its isoforms; eNOS (in vascularendothelial lining) and nNOS (in neurons), and the inducible variant(iNOS) found in macrophages, white blood cells, fibroblasts, endothelialcells, and keratinocytes. The function of NO may differ with itscellular source; fibroblast NO supports collagen synthesis, whileendothelial NO affects angiogenesis, and macrophage NO is cytostatic tobacteria (Rohdewald, P. and Ferrari V., Patent Application US2004137081(2004). On the other hand, arginase shares and competes on the naturalsubstrate of NOS, namely; L-Arginine. L-hydroxyarginine and nitrite, theintermediate and end product, respectively, of the NO pathway, are bothstrong arginase inhibitors (Hrabak, A. et al., FEBS Lett. 390:203-206(1996)). Conversely, urea, the end product of arginase activity,inhibits NO formation and NO-dependent processes (Witte, M. B. andBarbul. A., Wound Rep. Reg. 11:419-423 (2003)).

Arginine in cardiovascular conditions: Arginine proved to be beneficialif administrated to patients with cardiovascular conditions in numerousclinical trials (reviewed by Appleton, J., Altern. Med. Rev. 7:512-522(2002)). For example, oral arginine supplementation dramaticallyimproved exercise capacity and tolerance in patients with anginapectoris (Bednarz, B. et al., Int. J. Cardiol. 75:205-210 (2000)), andsignificantly improved their blood flow, arterial compliance, and renalfunction in cases with congestive heart failure (CHF) (Watanabe, G. H.et al., J. Hypertens. 18:229-234 (2000)). Animal studies suggestedanti-atherogenic effects of supplemental arginine, including improvedvasodilatation, inhibition of plaque formation, decreased thickening ofthe aortic tunica intima, and a normalized platelet aggregation inhypercholesterolemic human adults (Nakaki, T. and Kato R., Jpn J.Pharmacol. 66:167-171 (1994)). Moreover, early provision of arginineimproved hypertension, prevented renal failure in rats and humans(Sanders, P. W., Am. J. Kidney Dis. 28:775-782 (1996)), and enhanced theresponse of hypertensive patients to medicaments such as enalapril(Pezza, V. et al., Am. J. Hypertens. 11:1267-1270 (1998)). Additionally,arginine significantly improved symptoms of intermittent claudication(Böger, R. H. et al., J. Am. Coll. Cardiol. 32:1336-1344 (1998)), and ofpreeclampsia (Roberts, J. M., Am. J. Kidney Dis. 33:992-997 (1999)).

Arginine in growth hormone (GH) secretion and athletic performance:Growth hormone is responsible for enhancing muscle growth, burning fatand maintaining the immune system, however, its secretion begins todecline in the human body by the age of thirty (reviewed by Dean, W. andPryor, K., Growth hormone: amino acids as GH secretagogues—a review ofthe literature. Vit. Res. News; available at www.vrp.com (2001)).Although the mechanism is not well understood, arginine is known toenhance GH secretion. Furthermore, clinicians routinely use an arginineinfusion test to determine the responsiveness of the pituitary gland toreleasing GH in humans (Penny, R. et al., J. Clin. Endocrinol.29:1499-1501 (1969)). Low dose intravenous (IV) infusion of arginine wasassociated with a 52% rise in serum arginine and a significant increasein serum GH levels. On the other hand, oral arginine, unlike IVarginine, was suggested to be ineffective means of enhancing GHsecretion (Marcell, T. J. et al., J. Gerontol. 54:395-399 (1999)), whilehigh doses of oral arginine aspartate were suggested to act as a growthhormone secretagogue only at night (Besset, A. et al., Acta Endocrinol.99:18-23 (1982)). In fish, arginine is an essential amino acid, thus,dietary arginine is essential for optimum growth and efficient feedutilization and its deficiency causes reduced growth rate, loweredimmune response, and increased mortality (Ahmed, I. and Khan, M. A.,Aquacult. Nutr. 10:217-225 (2004)).

Arginine in wound, burns, critical trauma, and senile dementia: Becausearginine is intimately involved in cell signaling through the productionof nitric oxide and with cell proliferation through its metabolism toornithine and the other polyamines, numerous studies showed that itssupplementation is essential for healing. This effect is not dependanton the route of administration and is supposed to be associated with thesynthesis pathways of collagen, NO, ornithine, and polyamines (Witte, M.B. and Barbul. A., Wound Rep. Reg. 11:419-423 (2003)).

Collagen synthesis is essential for scar formation, which is the basisfor most mammalian healing. Rats fed arginine-free diet showed impairedwound healing while humans and animals fed arginine-enriched diet hadimproved collagen deposition and wound breaking strength (Barbul, A. etal., Surgery 108:331-336 (1990)). Arginine effect on collagen synthesisis supposed to be mediated in part via NO synthesis because iNOSinhibitors decreased collagen deposition and retarded healing ofincisional wounds, whereas higher levels of NO metabolites were found inwound fluids after arginine-supplementation (Murrell, G. A. C. et al.,Inflamm. Res. 46:19-27 (1997); Schäffer, M. R. et al., Eur. J. Surg.165:262-267 (1999)). Effects of NO on wound healing are even suggestedto be systemically mediated (Witte, M. B. and Barbul. A., Wound Rep.Reg. 11:419-423 (2003)) because: 1) arginine-free nutrition inhibits theinduced NO synthesis in several organs not only at the wound site; 2) NOmediates inflammation-induced edema and inhibits cell infiltration intogranulomas; 3) the effect of NO on wound healing is not onlyiNOS-mediated since eNOS knock-out mice also show impaired healing; and4) iNOS inhibitors have a high lethality in high concentrations.Additionally, while wound contraction contributes largely to the closureof open wounds, excisional wounds closure is delayed by iNOS inhibition(Stallmeyer, B. et al., J. Invest. Dermatol. 113:1090-1098 (1999)) andiNOS knock-out mice show delayed closure of excisional wounds that canbe reversed by transfection with iNOS-cDNA (Yamasaki, K. et al., J.Clin. Invest. 101:967-971 (1998)). All this data led to believing thatarginine metabolism via NOS is essential for the positive effects ofarginine on healing (Shi, H. P. et al., Surgery 128:374-378 (2000)).

Induction or overexpression of arginase, which represents the first stepin polyamine biosynthesis, enhances endothelial cell proliferation (Wei,L. H. et al., Proc. Natl. Acad. Sci. USA 98:9260-9264 (2001); Witte, M.B. and Barbul. A., Wound Rep. Reg. 11:419-423 (2003)). Arginine is alsoknown to enhance wound healing by stimulating host and wound T-cellresponses, which then increase fibroblastic responses (Barbul, A. etal., Surgery 108:331-336 (1990)). In healthy humans, arginine enhancesthe mitogenic activity of peripheral blood lymphocytes and greatlyreduces post-traumatic impairment in lymphocyte blastogenesis (Daly, J.M. et al., Ann. Surg. 208:512-23 (1988)). Arginine has been shown to becritical for bone marrow lymphocyte differentiation. BecauseT-lymphocytes are essential for normal wound healing, T-cell depletedmice and rats have a significantly impaired wound healing. Other studiesshowed that the beneficial effects of supplemental arginine on woundhealing are similar to the effects of administered GH to wounded animalsor burned children (Jorgensen, P. H and Andreassen, T. T., Acta Chir.Scand. 154:623-626 (1988); Herndon, D. N. et al., Ann. Surg. 212:424-9(1990)), which is due to the well-known high secretagogue activity ofarginine on the pituitary and pancreatic glands. This was confirmed bytests on hypophysectomized animals where arginine did not affect woundhealing (Wei, L. H. et al., Proc. Natl. Acad. Sci. USA 98:9260-9264(2001); Witte, M. B. and Barbul. A., Wound Rep. Reg. 11:419-423 (2003)).

Seifter, E. et al., Surgery 84:224-230 (1978) showed that argininebecomes essential in post-traumatic situations; arginine-deficient ratssubjected to minor trauma showed significantly more weight loss andmortalities. Also burn injuries significantly increase arginineoxidation and fluctuation in its reserves. The often used totalparenteral nutrition (TPN) increases conversion of arginine to ornithineand proportionally increases irreversible arginine oxidation. Elevatedarginine oxidation, coupled with limited de novo synthesis, makearginine conditionally essential in severely burned patients receivingTPN (Yu, Y. M. et al., Am. J. Physiol. Endocrinol. Metab. 280:E509-E517(2001)). Several other studies demonstrated that arginine reduces lengthof hospital stay, acquired infections, immune impairment among burn andtrauma patients (Appleton, J., Altern. Med. Rev. 7:512-522 (2002)), andlipid peroxidation in elderly patients with senile dementia (Ohtsuka, Y.and Nakaya J., Am. J. Med. 1:108-439 (2000)). These numerousobservations, coupled with relative safety, made the use of argininevery attractive for the care of traumatized, burned, or seriously illpatients (Witte, M. B. and Barbul. A., Wound Rep. Reg. 11:419-423(2003)).

Arginine in immunomodulation and cancer: Arginine is a potentimmunomodulator and was shown to have beneficial effects in catabolicconditions such as sepsis and postoperative stress (Evoy, D. et al.Nutrition 14:611-617 (1998); Appleton, J., Altern. Med. Rev. 7:512-522(2002)). Arginine may also be of benefit in individuals with HIV/AIDS.The combination of glutamine, arginine, and HMB (hydroxymethylbutyrate)prevented loss of lean body mass in individuals with AIDS (Swanson, B.,Nutrition 18:688-690 (2002)). Animal and human trials showed that largedoses of arginine may interfere with tumor induction and that short-termsupplementation with large doses of arginine assists in maintaining ofthe immune functions during chemotherapy (Appleton, J., Altern. Med.Rev. 7:512-522 (2002)). Arginine in diabetes and insulin resistance:Reduced plasma arginine level and impaired endothelium-dependentrelaxation are observed in humans and animals with diabetes mellitus(DM). Endothelial NO deficiency was supposed to be a likely reason forthis. Therefore, arginine supplementation was suggested to improve theseconditions; IV arginine reduced blood pressure and platelet aggregationin patients with DM type 1 (Giugliano, D. et al., Am. J. Physiol.273:E606-E612 (1997)), while low-dose IV arginine improved insulinsensitivity in obese and type 2 DM patients as well as in healthysubjects (Wascher, T. C. et al., Eur. J. Clin. Invest. 27:690-695(1997)). Arginine may also counteract lipid peroxidation and therebyreduce microangiopathic long-term complications of DM. Moreover, adouble-blind trial showed that oral arginine supplementationsignificantly improved peripheral and hepatic insulin sensitivity inpatients with type 2 DM (Appleton, J., Altern. Med. Rev. 7:512-522(2002)).

Arginine in gastrointestinal conditions: The action of arginine on NO,gastrin, and polyamines which exerts its hyperemic, angiogenic, andgrowth-promoting effects were associated with an acceleration in ulcerhealing during preliminary studies (Brzozowski, T., J. Gastroenterol.32:442-452 (1997)). Additionally, NO plays an important role in theregulation of gastrointestinal motility. Oral arginine supplementationsignificantly decreased the frequency and intensity of chest painattacks in patients with esophageal motility disorders (Appleton, J.,Altern. Med. Rev. 7:512-522 (2002)). Similarly, the L-Arginine-NOpathway is involved in the regulation of gallbladder motility andL-Arginine ingestion increased fasting and residual gallbladder volumes(Luiking, Y. C. et al., Am. J. Physiol. 274:984-991 (1998)). Arginine ingenitourinary conditions: A survey carried out in the United States(1999) indicated that 31% of men and 43% of women aged 18 to 59 yearshave varying degrees of sexual dysfunction (Christianson, D. W., Acc.Chem. Res. 38:191-201 (2005)). This problem can have physiological orpsychological reasons or both. In men, sexual dysfunction is brieflydescribed as erectile dysfunction (impotence or ED), whereas in women,sexual dysfunction is classified in four main categories: hypoactivesexual desire, orgasmic disorder, sexual pain disorder, and sexualarousal disorder (Basson, R. et al., J. Urol. 163:888-893 (2000)). Thelatter, defined as the inability to achieve or maintain sufficientsexual excitement including clitoral erection and genital engorgement,is analogous to male ED in being caused by deficiency in genital bloodcirculation. This can result in both genders from physiological defectsin the enzyme-catalyzed reactions governing blood flow into and out ofthe corpus cavernosum, a muscularized chamber of expandable tissue thatbecomes engorged with blood in the erect penis or clitoris(Christianson, D. W., Acc. Chem. Res. 38:191-201 (2005)).

Penile erection, in particular, is a hemodynamic process involvingincreased arterial inflow and restricted venous outflow followingcentral or peripheral sexual stimulation (Musicki, B. et al., Biol.Reprod. 70:282-289 (2004)). The involvement of NO, generated by bothendothelial NO synthase (eNOS) and the neuronal penile NO synthase(PnNOS) as the main mediator of penile erection, is well documented. NOdiffuses to the adjacent target smooth muscle tissue stimulatingguanylylcyclase to produce cyclic guanosine monophosphate (cGMP) leadingto relaxation of the corpora cavernosa (Ferrini, M. et al., Biol.Reprod. 64:974-982 (2001)). Conversely, erection is terminated whencGMP-specific phosphodiesterases (PDEs) hydrolyze cGMP to 5′-GMP leadingto smooth muscle contraction (Firoozi, F. et al., Br. J. Urol. Int.96:164-168 (2005)). Thus, L-Arginine and drugs acting on theL-Arginine-NO pathway are attractive as therapeutics for ED. Moreover,selective inhibitors of PDEs as Sildenafil citrate (Viagra®), whichinhibit cGMP degradation and thereby prolong erection, are widelyapplied. However, Sildenafil has a systemic vasodilating and hypotensiveeffect leading to a broad range of side effects starting from headaches,and may even reach death (Cohen, J. S., Ann. Pharmaco. Ther. 35:337-342(2001)).

L-Arginine “nutraceuticals” are often explored as remedies for male andfemale sexual arousal. For example; long term or supplementation withsupra-physiologic doses of dietary L-Arginine enhanced intracavernosalpressure and erectile function in rat (Moody et al. 1997), and men,respectively. Long term L-Arginine supplementation improved ED in menwith abnormal nitric oxide metabolism (Zorgniotti and Lizza 1994), whilein another study on women, L-Arginine nutritional supplement improvedsatisfaction with overall sexual life in 73.5% of the tested subjects(Ito, T. Y. et al., J. Sex Marital Ther. 27:541-549 (2001)). On theother hand, NOS is not the only enzyme affecting penile erection,arginase shares its sole substrate arginine and is coexpressed with NOSin smooth muscle tissue in male and female genitalia. Therefore,arginase inhibition may enhance the NO-dependent physiological processesrequired for sexual arousal. Because many arginase inhibitors have noapparent effect on systemic arterial blood pressure, it became anotherpotential target for the treatment of sexual dysfunction (Christianson,D. W., Acc. Chem. Res. 38:191-201 (2005)).

Arginine in infertility and pregnancy: Arginine is required for normalspermatogenesis in men (Appleton, J., Altern. Med. Rev. 7:512-522(2002)). Over 50 years ago, researchers found that feeding adult men onarginine deficient diet for nine days decreased sperm counts by 90% andincreased the percentage of non-motile sperms approximately 10-folds(Holt, L. E. Jr. and Albanese, A. A., Trans. Assoc. Am. Physicians58:143-156 (1944)). Oral administration of 0.5 g arginine-HCl per day toinfertile men for several weeks markedly increased sperm counts andmotility in a majority of tested patients, and resulted in successfulpregnancies (Tanimura, J., Bull. Osaka Med. School 13:84-89 (1967)).Similar effects on oligospermia and conception rates have been reportedin other preliminary trials (Tanimura, J., Bull. Osaka Med. School13:84-89 (1967); De-Aloysio, D. et al., Acta Eur. Fertil. 13:133-167(1982)) and improved fertility. However, when baseline sperm counts wereless than 10 million/ml, arginine supplementation could not help(Mroueh, A., Fertil. Steril. 21:217-219 (1970); Appleton, J., Altern.Med. Rev. 7:512-522 (2002)).

Oral arginine supplementation for women poorly responding to in vitrofertilization improved ovarian response, endometrial receptivity, andpregnancy rate in one study (Battaglia, C. et al., Hum. Reprod.14:1690-1697 (1999)). Additionally, intravenous arginine infusion (30 gover 30 min) in women with premature uterine contractions, transientlyreduced uterine contractility (Facchinetti, F. et al., J. Perinat. Med.24:283-285 (1996)). Further evidence from human and animal studiesindicated that nitric oxide inhibits uterine contractility duringpregnancy and may help and thereby acting against preterm labor anddelivery (Appleton, J., Altern. Med. Rev. 7:512-522 (2002)).

In patients with interstitial cystitis (IC), oral arginine over sixmonths significantly decreased urinary voiding discomfort, abdominalpain, and vaginal/urethral pain. Urinary frequency during day and nightwas also significantly decreased (Smith, S. D. et al., J. Urol.158:703-708 (1997); Appleton, J., Altern. Med. Rev. 7:512-522 (2002)).

L-Lysine: L-Lysine is an essential basic amino acid, has a molecularweight of 146.19 g/mol, and carries a positive charge at physiologicalpH. While the D-stereoisomer of lysine is not biologically active,L-Lysine is a known food additive for human and animal (Cynober, L. A.Metabolic and therapeutic aspects of amino acids in clinical nutrition.2nd ed. CRC Press LLC, Boca Raton, USA (2003)). Ingested L-Lysine isabsorbed from the lumen of the small intestine into the enterocytes byactive transport. A portion thereof is metabolized within theenterocytes and the rest is transported via the portal circulation tothe liver to participate in protein biosynthesis or to be metabolized toL-alpha-aminoadipic acid semialdehyde, which is further metabolized toacetoacetyl-CoA. L-Lysine that is not metabolized in the liver istransported to the various tissues of the body (Cynober, L. A. Metabolicand therapeutic aspects of amino acids in clinical nutrition. 2nd ed.CRC Press LLC, Boca Raton, USA (2003)).

Lysine has many functions. It serves as a precursor for glycogen,glucose and lipids or it serves directly for energy production. It isconcentrated in muscles, promotes bone growth, and enhances theformation of collagen (Voet, D. and Voet, J. G., Biochemistry. 3th ed.John Wiley and Sons Inc., New York (2004)). Collagen is the basic matrixof the connective tissues (see before), skin, cartilage, and bone.Lysine deficiency may contribute to reduced growth and immunity,impaired sperm health, along with an increase in urinary calcium. Thislatter fact suggested that adequate lysine may help prevent osteoporosisthrough better absorption and deposition of calcium (Flodin, N. W., J.Am. Coll. Nutr. 16:7-21 (1997)). L-Lysine became popular as anutritional supplement when some studies showed that it can decrease therecurrence rate of herpes simplex infections and in stimulating growthhormone secretion (see below).

Recommended dosages, side effects, and contraindications: Supplementaldoses of the above discussed amino acids are largely variable dependingon the conditions to be treated. However, the normal dietary needs ofthe essential amino acid lysine for average human are estimated to be0.75-1 g daily to avoid deficiency problems. Doses of arginine used inclinical research have varied considerably, from as little as 0.5 g perday for oligospermia to as much as 30 g per day for cancer,preeclampsia, and premature uterine contractions. Significant adverseeffects have not been reported on the supplementation of the discussedamino acids throughout this article. However, many of the mentionedclinical applications need to be confirmed with more controlled as wellas long-term studies. This article summarizes only the positive reportson the effects of these amino acids, whereas, contra-reports also existwhere many of these effects could not be confirmed (for complete reviewssee Flodin, N. W., J. Am. Coll. Nutr. 16:7-21 (1997); Appleton, J.,Altern. Med. Rev. 7:512-522 (2002); and Dean, W. and Pryor, K., Growthhormone: amino acids as GH secretagogues—a review of the literature.Vit. Res. News; available at www.vrp.com (2001)).

Dipeptides and mixtures of aspartate, arginine and lysine in clinicaltherapy: Aspartate and arginine are favorably administrated together asdipeptides or in mixtures to provide higher bioavailability for bothamino acids and thereby increase their effectiveness in lower doses. Theadministration of both amino acids was investigated during severalstudies for the treatment of different physiological disorders. Ingeneral, both amino acids can be administrated in the dipeptide form forall the above-mentioned applications for the free amino acids. Thefollowing section summarizes though, the research results reportedspecifically on the combined administration form. These reports emergedfrom studies on wound therapy, on endocrine conditions as the GHsecretary disorders and enhancing athletic performance, or ongenitourinary conditions including erectile dysfunction and male andfemale infertility.

Arginine-aspartate was tested clinically in 1965 for the first timeagainst physical and psychic asthenia (Duruy, A. and Baujat, J. P., Vie.Med. Int. 9:1589 (1965)) and the positive effect was later confirmed(Duruy, A., Med. Int. 1:203 (1966)). Other studies showed that long-termadministration of arginine-aspartate improves aerobic energy metabolismand performance (Sellier, J., Rev. Med. Toulouse 5:879 (1979); Schmid,P. et al., Leistungssport 10:486-495 (1980)). Arginine-aspartatesupplementation enhanced wound healing and the immune functions ofT-cells (Barbul, A. et al., Surgery 108:331-336 (1990)). Other positiveeffects were also reported on athletic performance; for example on lipidmetabolism, where arginine intake for only 2 weeks caused sinking in thetotal cholesterol concentrations (Hurson, M. et al., J. Parenter.Enteral Nutr. 19:227-230 (1995)). Thus, L-Arginine-L-Aspartate(Sargenor®) is widely used by athletes and patients to increase trainingeffects as well as exercise tolerance. Impressive effects on enduringperformance in this field have been reported after prolonged intake ofL-Arginine-L-Aspartate causing decreased blood lactate concentrationsand heart rates during submaximal exercise and increased oxygen uptakewith workload increments (Schmid, P. et al., Leistungssport 10:486-495(1980); Sellier, J., Rev. Med. Toulouse 5:879 (1979); Burtscher, M. etal., J. Sports. Sci. Med. 4:314-322 (2005)).

Dietary supplementation with 30 g/d arginine aspartate for 2 weeks tohealthy elderly human volunteers enhanced wound collagen accumulationsignificantly (Witte, M. B. and Barbul. A., Wound Rep. Reg. 11:419-423(2003)). When 250 mg/kg/day of oral arginine aspartate wereadministrated to five healthy subjects aged 20 to 35 for seven days, a60% rise in GH occurred during slow wave sleep (Besset, A. et al., ActaEndocrinol. 99:18-23 (1982)). Another group of researchers achievedpromising results after treating 12 normal adults with one large dose(37.5 g) of oral arginine aspartate, which caused small but significantrelease of serum hGH (Elsair 1987). This made arginine aspartateinteresting for body builders wishing to take advantage of the anabolicproperties of the hGH (Macintyre, J. G., Sports Med. 4:129-142 (1987)).

Orally administrated L-Arginine-L-Aspartate was also reported to inducepositive effects in treatment of some types of cancer. For example itinduced antimetastatic effects on salivary adenoid cystic carcinoma inmice, accompanied by inhibited pulmonary metastatic foci formation andprolonged survival. Further in vitro and in vivo experiments confirmedthese results (Li, F. et al., Chin. J. Stomatol. 36:464-466 (2001); Li,F. et al., Chin. J. Stomatol. 37:87-89 (2002); Appleton, J., Altern.Med. Rev. 7:512-522 (2002)).

In the field of dental health, plaque, the closely adhering spongyorganic material on teeth surfaces, was found to accept peptides ofcertain size and shape within its matrix. Furthermore, peptides of 2-4amino acid units, one or more of which is arginine, were shown to bestored in plaque protected from dilution and to effectively restoremouth pH to a non-carious level (6.1 or higher). It was further shownthat these oligomers are effective even when provided simultaneouslywith carbohydrates. This suggested the inclusion of such peptides incommon dental products such as toothpastes and chewing gum (Kleinberg,I., U.S. Pat. No. 4,225,579 (1980)).

L-Arginine-L-Aspartate was also applied for the treatment ofgenitourinary disorders. ED is common in 25% of males aged 45-70 yearswith moderate erectile dysfunction and in 10% with severe erectiledysfunction (Kernohan, A. F. B. et al., Br. J. Clin. Pharmacol. 59:85-93(2004)). Recently, “L-arginyl aspartate” was used as a component ofseveral pharmaceuticals for the treatment of male ED such as Prelox®(Lamm, S. et al., Eur. Bull. Drug Res. 11:29-37 (2003)). Clinicalstudies on the components of Prelox® showed improved erectile functionin 5% and 92% of forty men with ED after receiving 3 doses of 1 g of“L-arginyl aspartate” (Sargenor®) (1.7 g arginine daily) alone, ortogether with Pycnogenol® (stimulates NOS secretion), respectively(Stanislavov, R. and Nikolova, V., J. Sex Marit. Ther. 29:207-213(2003)). During a long term study, fifty men aged between 45 and 60having ED, lowered volume of semen, reduced sperm motility andmorphological abnormalities of sperms, were treated first with Sargenor®alone for 1 month. 10% of these men experienced normal erection. Afteraddition of Pycnogenol® to the treatment for the second month, thepercentage of men with normal erection increased to 80%. The treatmentwas continued for a period of one year during which sperm quality wassignificantly improved and 42% of the couples achieved pregnancy(Stanislavov, R. and Nikolova, V., Int. J. Impot. Res. 14(4):565 (2002);Lamm, S. et al., Eur. Bull. Drug Res. 11:29-37 (2003)). The laterobservation confirmed previous studies on the use of arginine-aspartatewhere several months supplementation increased sperm count and quality(Tanimura, J., Bull. Osaka Med. School 13:84-89 (1967); Schellen, T. M.and Declerq, J. A., Dermatol. Monatsschr. 164:578-80 (1978); De-Aloysio,D. et al., Acta Eur. Fertil. 13:133-167 (1982)) and improved fertility(Schacter, A. et al., J. Urol. 110:311-13 (1973); Schacter, A. et al.,Int. J. Gynaecol. Obstet. 11:206-209 (1973)).

Clinical reports on dipeptides consisting of aspartate and lysine hardlyexist because this dipeptide is not available in large amounts. However,this dipeptide form of lysine could be effective in the applicationfields known for free lysine or its salts (see before), in people withgenetic deficiency in lysine transporters, or simply as a food additive,with higher bioavailability than free lysine, for human and animals.Arginine and lysine work synergistically to release growth hormone (GH)(Suminski, R. R. et al., Int. J. Sport Nutr. 7:48-60 (1997)) and theirconcentrations are very important in human and animal nutrition (Ahmed,I. and Khan, M. A., Aquacult. Nutr. 10:217-225 (2004)). Low lysine:arginine ratios have hypocholesterolemic effect (Sanchez, A. et al.,Nutr. 38:229-238 (1998)) and thus, patents were made for proteinmixtures with Arg:Lys ratio of at least 5.5: 1 to be used for patientswith cardiovascular diseases (Radha, C. et al., U.S. Pat. No. 7,091,001(2006)). In contrast, and because proteins of herpes simplex virus arerich in L-Arginine, high lysine to arginine ratio in the diet is knownto help reducing viral replication, healing times, and thecytopathogenicity during outbreaks (Griffith, R. S. et al.,Dermatologica 156(5): 257-267 (1978)). Thus, in herpes prevention andtreatment, avoiding arginine-rich foods and eating more lysine-richfoods is suggested to be helpful.

Recent research has suggested that therapy using L-Lysine and L-Argininetogether is useful and possibly even better than the arginine/ornithinecombination in stimulating hGH, and thereby improving muscle building,weight gain, and immune support. In 15 healthy male subjects aged 15 to20 years old, 1.2 g of arginine pyroglutamate combined with L-Lysinehydrochloride significantly elevated GH levels from two to eight timesafter consuming the amino acid mixture (Isidori, A. et al., Curr. Med.Res. Opin. 7:475-481 (1981)). Another study indicated that ingestion of1.5 g arginine and 1.5 g lysine ingested under resting conditions causesan acute increase in GH secretion (Suminski, R. R. et al., Int. J. SportNutr. 7:48-60 (1997)).

To simulate the natural conditions inside the digestive tracts, theprepared gut “juice” was used as inoculum and as nutritional supplementin anaerobic Hungate tubes containing CGP. The complete degradation ofCGP in all tubes indicates that CGP can be easily degraded in suchanaerobic milieu like that of the digestive tracts. This was alsoconfirmed by the short degradation periods which are analogous to thoseobserved previously for strict and facultative anaerobic CGP degradingbacteria (Obst, M. et al., Appl. Environ. Microbiol. 71:3642-3652(2005); Sallam, A. et al., Submitted for publication (2008)).

During this study, and for the first time the wide spread of CGPdegrading bacteria in animal, avian and fish digestive tracts isdemonstrated (Table 1, 2). The morphological diversity between the CGPdegrading colonies observed during purification procedures and laterbetween the resulting axenic culture was also shown by previous studieson environmental samples, which showed wide spread of CGP degradersamong prokaryotes (Table 2). On the other hand, the capability todegrade CGP seems to be more spread among species of particular genera;investigations on extracellular CGP degradation until today show thatCGP degraders are wide spread among the genera Pseudomonas and Bacillus(Obst, M. et al., J. Biol. Chem. 277:25096-25105 (2002); Obst, M. etal., Biomacromolecules 5:153-161 (2004); Sallam, A. et al., Submittedfor publication (2008), this study). However, this might be associatedwith the applied laboratory conditions which might have favorized thesebacteria or simply because of the predominance of these bacterial generain nature. Additionally, many CGP degrading bacteria, unlike strains ofgenera Pseudomonas and Bacillus, are extremely hard to be brought toaxenic cultures without loosing the ability to degrade CGP (Obst, M. etal., Appl. Environ. Microbiol. 71:3642-3652 (2005); Krug, A., Diplomthesis, Institut für Molekulare Mikrobiologie and Biotechnologie,Westfälische Wilhelms-Universität, Münster, Germany (2001)) and areusually disregarded.

Partial CGP degradation was observed only for axenic cultures, underanaerobic conditions, and for only 8 strains from 46 strains thatdegraded CGP anaerobically. A plausible reason for this is the lack ofnatural interactions between these strains and others as in theirnatural milieu, which leads often to the accumulation or exhaustion oflimiting or necessary substances, respectively. This is in accordancewith previous observations on the strict anaerobic endospore former S.hongkongensis strain KI where growth and CGP utilization were largelyenhanced in the presence of its co-isolate Citrobacter amalonaticusstrain G (Obst, M. et al., Appl. Environ. Microbiol. 71:3642-3652(2005)).

Among the eight identified isolates, several strains showed capabilityof anaerobic CGP degradation although they belong to genera known to beaerobic like Bacillus or Pseudomonas. However, strains of B. subtilisand B. megaterium are known to grow anaerobically and some are known toreduce nitrate (Glaser, P. et al., J. Bacteriol. 177:1112-1115 (1995)).Similarly, anaerobic growth and nitrate reduction of members of genusPseudomonas is well investigated (Sallam, A. et al., Submitted forpublication (2008)). Species of Micromonospora, Streptomyces andBrevibacillus are also known to be facultative anaerobic (Cochrane, V.W., Annu. Rev. Microbiol. 15:1-26 (1961); Borodina, I. et al., GenomeRes. 15:820-829 (2005); Baek, S. H. et al., Int. J. Syst. Evol.Microbiol. 56:2665-2669 (2006)). In general, the recent investigationson CGP degradation point toward a wide spread of CGP degrading bacteriaamong the facultative anaerobes.

Although CGP and its dipeptides are simple proteinacious substances ofnatural origin, clinical studies are required to bring these substancesto the market. The wide spread of CGP degrading bacteria in gut flora ofeach of the mammalian, avian or fish tested during this work, providesthe first evidence that orally administrated CGP would be fast andeasily degraded at least microbially. The isolation of such bacteriafrom the lower digestive tract of different animals and birds (Table 1)indicates that if the digestion of CGP was not completed in the upperdigestive tract, it would possibly continue in the lower part.

HPLC analysis reveled that dipeptides were the degradation products ofCGP by all 62 isolates. No dipeptide oligomers of higher order like(β-Asp-Arg)₂ tetrapeptides were produced from CGP. This is in accordancewith the effect of the CGPases from P. anguilliseptica strain BI (Obst,M. et al., J. Biol. Chem. 277:25096-25105 (2002), S. hongkongensisstrain KI (Obst, M. et al., Appl. Environ. Microbiol. 71:3642-3652(2005)) and P. alcaligenes strain DIP1 (Sallam, A. et al., Submitted forpublication (2008)). Only in case of Bacillus megaterium strain BAC19,such oligomers of higher order were detected (Obst, M. et al.,Biomacromolecules 5:153-161 (2004)). Spirulina is known for centuriesfor its nutritional and therapeutic effects and is consumed as food inmany countries until today. The protein content of Spirulina is known toreach over 60% (Narasimha, D. L. R. et al., J. Sci. Food Agric.33:456-460 (1982)). The presence of CGP in market-available products ofSpirulina platensis indicates that CGP might participate in thewell-being effect of regular consumption of Spirulina. However, thedetermined CGP contents in the analyzed samples varied largely and wererelatively low. This is in agreement with previous studies on CGPaccumulation in cyanobacteria, where the accumulation of CGP is known tobe affected by many factors and to vary accordingly (Elbahloul, Y. etal., Appl. Environ. Microbiol. 71:7759-7767 (2005)). Moreover, themarket available Spirulina products surely have gone through numerousclinical and toxicological tests before they were licensed forcommercialization. This indicates that extracted CGP and CGP-dipeptideswould induce no toxic effects if ingested orally. Moreover, CGPdipeptides are usually uptaken and utilized for growth in bacteria, andshowed no bacteriostatic or bactericidal effects during targetedinvestigations (data not shown). In general, the known effects andapplications of Spirulina are very similar to those proved for arginine,which suggests that a part of these effects may be actually due to itsarginine content (about 6%) including that of CGP.

Bacteria possess three peptide transport systems, two, oligopeptidepermease (Opp) and dipeptide permeases (Dpp), belonging to the largefamily of ABC (ATP-Binding Cassette) transporter and one for di- andtripeptides belonging to the PTR (Peptide TRansporter) family. Only thelatter system is conserved in higher eukaryotic organisms starting withyeast (Daniel, H. et al., Physiology 21:93-102 (2006)). In mammals, thecarrier system for di- and tripeptides PEPT (SLC15 family) includes 2variants; the intestinal PEPT1 (SLC15A1) and the renal isoform PEPT2(SLC15A2). They transport almost all possible di- and tripeptides instereoselective manner with a preference for L-α amino acids and theirderivatives. Peptides containing solely D- or four or more amino acidsare not accepted. Because PEPT1 has a prominent expression throughoutthe small intestine and due to its high transport capacity, all drugsubstrates of PEPT1 have an excellent oral availability, and thus, PEPT1has become a prime target for drug delivery (Daniel, H. et al.,Physiology 21:93-102 (2006)). The constituting L-amino acids of CGPdipeptides, Asp-Arg and Asp-Lys (recombinant CGP) are linked via α-βpeptide bonds. This type of bond and the stereo-structure of CGPdipeptides assume strongly that they would act as substrates for PEPTsystem, and can be transported from the lumen of the mammalian gut whenthey are ingested. Thus, the use of CGP and/or the dipeptides thereofwould be an ideal approach for the oral administration of theconstituting amino acids as therapeutic and/or nutritional agents.

The nutritional and the clinical values of amino acids are known forcenturies including those which constitute CGP such as aspartate,arginine and lysine and those which can be still integrated into itsstructure like citrulline, ornithine, canavanine or glutamate. Syntheticoligomer combinations of these amino acids were proven to have higherbioavailability than free amino acids and thus were often investigatedand applied in nutrition and therapy (see before). CGP represents anideal natural source for such oligomers which can be expected to be moreeffective in lower doses than their constituting amino acids in the freeform. Thus, absorption, safety, and the effect of CGP and its dipeptidesare currently under investigation. Moreover, studies on integratingother amino acids in CGP showed promising results (data not shown). Theresulting dipeptides could be applied in several therapeutic fields, forinstance, aspartate-ornithine in the treatment of liver diseases(Kircheis, G. et al., Hepatology 25:1351-1360 (1997)). Consequently, anyfuture alterations in CGP structure would increase the range of CGPdipeptides and subsequently extend their application scope astherapeutics and/or nutritional supplements.

A triphasic process was established for the large scale production ofβ-dipeptides from cyanophycin (CGP). Phase I is based on an optimizedacid extraction method for the technical isolation of CGP from biomass,a total of 704 g pure CGP were obtained and structurally containedaspartate, arginine, and little lysine. Phase II represents thefermentative production of an extracellular CGPase (CphE), the enzymewas produced from Pseudomonas alcaligenes strain DIP1 at 500 l scale andusing 1 g/l citrate as a sole substrate, 17.5 g crude protein powderwere gained and showed high degradation activity on CGP. Phase IIIcomprises the degradation of CGP via CphE, 250 g of CGP were degraded toβ-aspartate-arginine and β-aspartate-lysine dipeptides with a puritygrade of over 99% (TLC, HPLC). The overall efficiency of phase III was91%, while 78% (wt/wt) of the used CphE powder were recovered and showedsustained activity on CGP. The established process depends on materialsand equipments with industrial standards and applicable as it is forevery desired scale.

Strains of P. alcaligenes including strain DIP1 are known to grow on awide range of substrates and to require minimal amounts thereof. Thehigh enzyme productivity of such strains was also a main reason fortheir application in the fermentative production of extracellularlipases (WO 95/30744; Gerritse, G. et al., Appl. Environ. Microbiol.64:2644-2651 (1998); Moore, E. R. B. et al. Mai 1999. Pseudomonas:Nonmedical. In Moore et al. (ed.), The Prokaryotes: An EvolvingElectronic Resource for the Microbiological Community, 3^(rd) edition,release 3.0, Springer-Verlag, New York)). These characteristics, inaddition to the high stability and activity of the CGPase from strainDIP1 (Sallam, A., and A. Steinbüchel. 2007b. Anaerobic and aerobicdegradation of cyanophycin by the denitrifying bacterium Pseudomonasalcaligenes strain DIP1-Role of other three co-isolates in the mixedbacterial consortium. Submitted for publishing), were the main factorsto consider this strain ideal for the designed technical process.

Before the final triphasic process was created, several trials werecarried out to reach the same goal by cultivating strain DIP1 directlyon CGP, however, that strategy was less effective due to the fastconsume of CGP-dipeptides by the growing cells, this problem was avoidedduring the defined procedures of the triphasic process where cells ofstrain DIP1 are excluded. Furthermore, the dipeptide solutions obtainedby the direct cultivation have large volumes and therefore extremelyhard to handle, also the presence of small proteins and salts in theresulting dipeptide solutions represented another disadvantage of thatstrategy. In contrast, the degraded concentrations of CGP by thetriphasic process as well as the time for degradation are entirelycontrollable. Thereby, the out-coming dipeptide solutions are restrictedto small and easy to handle volumes. The highest tested concentration(50 g/l) is one of the aspects that can be still optimized for futureapplications rendering the process to be economically more effective.

Economic factors are generally of great importance for technicalprocesses, therefore, the obtained results during medium optimizationand substrate utilization were found satisfactory, citrate which is acheap substrate in technical quantities, and which was ideal for theapplied strain as a sole substrate, was previously applied for thefermentative production of extracellular lipases (Gerritse, G. et al.,Appl. Environ. Microbiol. 64:2644-2651 (1998)), however, the need for anoptimized medium for the production phase of crude CphE (phase II)emerged through the necessity of accurate monitoring of the turbiditygrade during fermentation, especially during the induction anddegradation phase. Previous experiences with experimentalCGP-degradation showed that unclear media as well as strong growth ofcells can be misleading, besides, better growth of cells did notessentially mean higher production of extracellular CGPases (unpublisheddata).

The CGP acid-extraction method of Frey, K. M. et al., Appl. Environ.Microbiol. 68: 3377-3384 (2002), which was successfully applied duringprevious studies, was optimized to become suitable for the isolation ofpure CGP from any technical amounts of biomass. The most effectivechange to the original method was the sterile-filtration step of thesolved CGP, this procedure guaranteed for the complete removal of anycell debris that are not soluble in diluted HCl. In contrast, theincreased purification steps, with diluted acid and water, may haveunnecessarily increased the loss of CGP, which explains the differencebetween the obtained CGP and the expected amount. The loss of CGP aswell as the time needed for its extraction can be minimized bycentrifugating CGP instead of leaving it to settle at 4° C.

Alternatively, the overall productivity of this extraction method can bedrastically increased if an effective instrument such as cross flow isapplied, in this case, 2 cassettes with 2 different COPs are required;one larger and the other smaller than the molecular size of the CGP tobe isolated, the 2 cassettes are to be applied for the alternatingfiltration of CGP in the solved and the precipitated states of CGP,respectively. However, the relatively high prices and the yet limitedlife of such ultrafiltration cassettes leave their application to be amatter of costs.

During the fermentative production of the crude CphE (phase II), theinduction with CGP was employed within the stationary phase to guaranteefor a maximum production of CphE, and to exclusively refer the turbiditychanges to the amount of CGP in the medium.

The pH of the medium increased to exceed the tolerated range (pH6.9-7.5) and was controlled by the addition of HCl, this is mostprobably due to the release of ammonia by cells of strain P. alcaligenesDIP1. A similar physiological behavior was also documented for thisstrain during previous investigations on CGP degradation (Sallam, A.,and A. Steinbüchel. 2007b. Anaerobic and aerobic degradation ofcyanophycin by the denitrifying bacterium Pseudomonas alcaligenes strainDIP1-Role of other three co-isolates in the mixed bacterial consortium.Submitted for publishing).

The identical purity grade of the resulting CGP-dipeptides, before andafter the different tested filtration systems, is clearly due to theinitial lack of impurities in the original dipeptide solution. Thisrepresents another advantage for applying a defined enzymatic process incomparison to the direct cultivation strategies with bacterial cells. Onthe other hand, the quantitative loss of dipeptides due to filtrationwas expected and unfortunately inevitable; several general factors areknown to cause such losses including; filter material, cut off points,and/or the characteristics of the filtered substance itself. Thisexplains also the loss of 9% of CGP and 22% of crude CphE during thedegradation phase (phase III). Besides, only new filter-membranes weretested (0.5-10 kDa. COPs), and thereby the lost amounts ofCGP-dipeptides most probably attached to the membranes surfaces untilsaturation.

Even though, the overall process affectivity of 91% is quite high,several aspects can be even more improved and thus are underoptimization, these aspects include higher productivity of crude CphE,possible technical purification strategies for CphE, and more effectiveconditions for the degradation phase (unpublished data). The commercialvalue of CGP-dipeptides are directly related to the production costs ofCGP itself, though, over the last few years, the production of CGP wasintensively investigated and optimized using several bacterial strains,thereby, mounting CGP contents are being achieved using new and moresuitable economic substrates, these developments seem to move fast inthe direction of commercial CGP production (see above), this way wasalso needed by other known bacterial poly(amino acids) such aspoly(glutamic acid) and poly(ε-lysine) to become commercialized fortechnical as well as food applications (reviewed by Oppermann-Sanio F.B. et al., Naturwissenschaften 89:11-22 (2002); Obst, M. et al.,Biomacromolecules 5:1166-1176 (2004)). Until then, the biomedical valueof CGP-dipeptides may in fact provide a balanced relation to the actualproduction costs of CGP. Therefore, the biomedical effects ofCGP-dipeptides are currently under investigation (Sallam, A., and A.Steinbüchel. 2007c. Potential of cyanophycin and its β-dipeptides aspossible additives in therapy, food and feed industries).

A previously applied biotechnological process for the large scaleproduction of β-dipeptides from cyanophycin (CGP) was optimized; theoriginal process consisted of three phases; phase I: large scaleextraction of pure CGP from biomass, phase II: large scale production ofcrude cyanophycinase (CphE_(al)) from Pseudomonas alcaligenes strainDIP1, phase III: CGP degradation to its dipeptides. Optimal cultivationconditions were determined for the second phase; 2 g l⁻¹ citrate, pH 6.5and cultivation temperature of 37° C. Optimal concentration for CGP asinductor for CphE_(al) was 50 g l⁻¹ which represents ⅕ of theconcentration applied previously. Maximum enzyme concentrations wereobtained 5 h after induction. The same concentration of CGP-dipeptidesshowed similar induction efficiency after only 3 h. Also an optimum of 4g l⁻¹ L-aspartate induced CphE_(al), however, with ⅓ efficiency comparedto CGP. CphE_(al) was purified via substrate-specific binding on CGP.The purified enzyme was characterized and came out to be a serineprotease with maximum activity at 50° C. and pH 7-8.5. Conditions forphase III of the original process (CGP degradation); 50 g CGP, 10 g l⁻¹crude CphE_(al) and incubation for 10 h at 30° C. could be optimized to;100 g l⁻¹ CGP, 10 g l⁻¹ crude CphE_(al) and incubation for only 4 h at50° C. CGP was degraded to β-aspartate-arginine and β-aspartate-lysinedipeptides with purity grade of over 99% (HPLC). These optimizationsrendered the technical process more cost, time and effort effective.Prior to the production of CGP on protamylasse at 500-l scalefermentation. Pre-tests on the available charge of protamylasse revealedthat 7% (vol/vol) was optimum for CGP production, however, during theprevious study of Elbahloul, Y. et al., Appl. Environ. Microbiol.71:7759-7767 (2005) on a former charge of protamylasse, the optimumconcentration was found to be 6% (vol/vol). This is due to thatprotamylasse, which is a residual compound of industrial starchproduction, is a complex medium with a composition that can vary fromcharge to charge and thus must be adjusted before application ascultivation medium.

During fermentation, and after the first 6 h, temperature was elevatedto 37° C. in order to inactivate the temperature-sensitive λ-repressor(c1857) and thereby enable the induction of CGP-synthetase (CphA) gene.At this time point, cells of recombinant E. coli strain were already fortwo h in the exponential growth phase. This fermentation course wasproved to be optimum for induction of the CGP-synthetase as well as formaximum intracellular accumulation of CGP (Frey, K. M. et al., Appl.Environ. Microbiol. 68:3377-3384 (2002)).

Also after 6 h of fermentation, turbidity of the medium showed a suddenjump with parallel increase in the automatically controlled stirring.This can be explained by the decrease of oxygen in the medium due tostrong cell growth in addition to the temperature elevation to 37° C.Obviously, oxygen content in the medium became lower than thepre-adjusted minimum and caused the automatic increase of stirring, thisin turn led to high formation of foam and air bubbles and subsequentlyto false OD_(850 nm) values. The manual addition of antifoam emulsioncaused a fast fall of turbidity to reach the normal level.

The fermentation was terminated when maximum CGP accumulation in thecells was microscopically estimated (after 15 h). However, lateranalysis of fermentation samples indicated that a better harvesting timewould have been after 13 h of incubation. At that time point, CGPcontent was about 13% (wt/wt of CDM) while after 15 h it declined to 10%(wt/wt of CDM). This lost was clearly due to the inaccuracy of themicroscopic estimation of CGP content which was the only possible methodduring fermentation. Moreover, the decrease of CGP content is mostprobably due to plasmid losses that occurred with time during incubation(Elbahloul, Y. et al., Appl. Environ. Microbiol. 71:7759-7767 (2005)).After extraction and purification of the polymer, HPLC analysis revealeda high purity grade for CGP and that it consists of the three aminoacids aspartate (47.7 mol %), arginine (45.6 mol %) and lysine (6.8 mol%). SDS-PAGE showed that CGP has a molecular size of 25-30 kDa. Thesecharacteristics are in strong agreement with those of previouslyproduced CGP by the same strain on several media (Frey, K. M. et al.,Appl. Environ. Microbiol. 68:3377-3384 (2002); Elbahloul, Y. et al.,Appl. Environ. Microbiol. 71:7759-7767 (2005)).

Recently, a simple mineral salt medium (SM-Medium) with 1 g l⁻¹ citratewas applied for the large scale production of CphE_(al) from P.alcaligenes strain DIP1. This medium was favorable due to its simple andcost-saving composition as well as due to its suitability for the usedstrain (Sallam, A., and A. Steinbüchel. 2008b. Biotechnological processfor the technical production of β-dipeptides from cyanophycin. Underpreparation). However, during this study, Even though the optimalcitrate concentration for growth was 6 g l⁻¹, no CphE_(al) was producedin such cultivations. Maximum production of CphE_(al) occurred incultures grown on 2 g citrate; this indicated that the enzyme is inducedonly under substrate limitation. Other experiments for temperature andpH optima indicated 37° C. and pH range of 5.5-7.5 with an optimum at6.5 to be optimal for strain DIP1 as well as for the production ofCphE_(al). This enhanced the efficiency of the production process whichwas initially applied on 1 g l⁻¹ citrate, pH 7.5 and an incubationtemperature of 30° C. (Sallam, A., and A. Steinbüchel. 2008b.Biotechnological process for the technical production of β-dipeptidesfrom cyanophycin. Under preparation).

Extended investigations on CphE_(al) induction revealed that only afifth (50 mg l⁻¹) of the previously applied CGP concentration (250 mgl⁻¹) was sufficient to render the same effect. Also CGP-dipeptides weresufficient in the same concentration (50 mg l⁻¹) to induce CphE_(al)with the same efficiency. However, the shorter incubation period (3 h)required until harvesting for cultures induced with dipeptides indicatesthat CGP-dipeptides are the actual inductors for CphE_(al) and not CGPitself. Additionally, aspartate was a successful inducer for CphE_(al),but other than CGP-dipeptides, a concentration of 4 g l⁻¹ and a furtherincubation period of 5 h were required for maximum CphE_(al) production,this represented a CphE_(al) production efficiency of only one third ofthat of CGP or the dipeptides thereof. Thus, choosing the inductor infuture applications remains case and cost dependant.

The third phase of the original process (large scale degradation of CGPvia crude CphE_(al)) was found to be much more effective at 50° C.instead of 30° C. This optimization rendered much higher concentrationsof CGP (up to 100 g l⁻¹) to be easily degradable in only a forth of thedegradation time at 30° C. This is in accordance with the Van 't Hoffequation where velocity of a reaction is doubled by a temperatureincrease of 10 kelvin (10° C.). Also volumes of degradation mixtures andthe risk of their contamination are minimized at such elevatedtemperature. At both incubation temperatures 30° C. and 50° C.,degradation time showed a collinear increase with decreasingconcentrations of crude CphE_(al) and with increasing CGPconcentrations. Thus, the resulting formula can be helpful to applyoptimum degradation parameters during future process applications.Because the efficiency of the degradation phase is much higher at 50°C., the formula was calculated for application at 50° C. and issubsequently suitable for CGP concentrations up to 100 g l⁻¹.

Organic solvents or ammonium sulfate precipitation provided weakpurification effect and low enzyme recovery rates. On the contrary, thedeveloped procedure by specific binding to CGP proved to be highlyeffective and has the advantage of separating CphE_(al) from otherproteins in crude solutions using one substance (CGP). The purificationmethod ends with the degradation of the CGP matrix to its dipeptides;these are in the same time the valuable end products of the process andthus can be directed further to the main production stream (no materialloss). The purification method is easy to up-scale and to be integratedin future process applications if desired.

Two formulas were created to increase the efficiency of the second phaseof the original process (large scale production of crude CphE_(al)) andthe possible purification thereof. The first formula (s.a.) is based onthe photometrical analysis of CphE_(al) and enables fast determinationof CphE_(al) content in crude supernatants. The determined concentrationof CphE_(al) can be then integrated into the second formula to estimatethe required amount of CGP to bind, and thereby purify, the completecontent of CphE_(al) in the supernatant. This scheme provides atrustable instrument for future production charges of crude CphE_(al)which might differ largely in their protein composition.

During experiments for substrate-specificity of the purified CphE_(al),not only CGP was degraded but also BSA, however, to lower extent. Thisindicates that the CGPase from P. alcaligenes strain DIP1 might be lessspecific than the previously characterized CphE_(Pa) and CphE_(Bm) fromP. anguilliseptica strain B1 and Bacillus megaterium strain BAC19,respectively. A more plausible explanation for this unspecific effect isthe presence of few other proteins in the purified enzyme solution. Eventhough these proteins were visible in SDS-PAGE only in highlyconcentrated samples and with intensive silver nitrate staining; only aminimum amount of a non-specific protease might produce such an effecton the tested substrates other than CGP.

CphE_(al) from P. alcaligenes strain DIP1 was largely inhibited by bothserine-protease inhibitors Pefabloc® and PMSF. This indicates that thisCGPase most probably belongs to serine-type proteases. This is inagreement with the results for the previously characterized CGPases;CphB, CphE_(Pa) and CphE_(Bm). Moreover, CphE_(al) was totally inhibitedby the tryptophan oxidizer N-bromosuccinimide that shows that atryptophan residue might be involved in the catalytic mechanism of theenzyme. Also this points out high similarity between CphE_(al) and theextracellular CphE_(Pa) and CphE_(Bm). CphE_(al) samples which weretreated with Leupeptin or EDTA showed no activity inhibition onCGP-overlay agar plates, however, HPLC analysis of samples treated withEDTA showed inhibition of the CGPase of about 75%. This is due toformation of large amounts of precipitates during OPA-derivatization andnot due to enzyme inhibition (Obst, M. et al., J. Biol. Chem.277:25096-25105 (2002); Obst, M. et al., Biomacromolecules 5:153-161(2004)). Biochemical characteristics of CphE_(al) of P. alcaligenesstrain DIP1 in comparison to the previously characterized CphB,CphE_(Pa) and CphE_(Bm) from Synechocystis sp. PCC6803, P.anguilliseptica strain B1 and Bacillus megaterium strain BAC19,respectively, are demonstrated in table 3. Although severalcharacteristics of the purified CphE_(al) were relatively similar tothose from CphE_(Pa) and CphE_(Bm), some relevant differences can beobserved such as molecular size and optimum temperature. The latter was50° C. for CphE_(al), which is the highest temperature optimum for allknown CGPases and thereby provides a great benefit for applying thisenzyme in the pure as well as in the crude form. The purified enzymeshowed an optimum pH range of 7-8.5 with an optimum of 8.5 which shiftsfrom that determined for the crude enzyme (5.5-7.5 with an optimum of6.5). This is most probably due to the presence of many other proteinsin the crude extract representing a complex milieu; interactions withinsuch milieu may in turn affect the structure and/or properties of theCGPase. The cell line Pseudomonas alcaligenes DIP1 was deposited on Jun.10, 2008 at the DMSZ, Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany underdeposition No. DSM 21533.

The invention is further described in the following examples, which arehowever, not to be construed as limiting the invention.

EXAMPLES Materials and Methods

Sampling and samples preparation: The gut flora samples were collectedfrom freshly slaughtered healthy exemplars (Table 1). Except forruminants (feeding history could not be determined), all chosen animals,birds and fishes were at least partially free living or free grazing.Samples were collected from several sites (Table 1) along the digestivetract of each source animal by completely filling 50 ml sterile falcontubes, which were kept at 4° C. until use. Samples were diluted(Table 1) with sterile normal saline and then filtered (folded filter,Schleicher & Schuell, Dassel, Germany) from solid materials understerile conditions. Faecal samples were chopped in sterile saline andfiltered as described above.

Media: For enrichment of bacteria capable of CGP degradation underanaerobic conditions, the following basal medium (BM) was applied: 1.0 gNH₄Cl, 3.0 g KH₂PO₄, 3.0 g K₂HPO₄, 0.1 g KCl, 0.5 g MgCl₂.6H₂O, 0.1 gCaCl₂.2H₂O, 0.5 g NaCl, 0.5 g cysteine-HCl, 0.5 g yeast extract (unlessotherwise indicated in text), 10 ml of SL10 trace elements solution, and1 mg of resazurin per I of distilled water. pH was adjusted to 7.0 withKOH. The medium was boiled, and after Na₂S.9H₂O had been added to afinal concentration of 0.04% (wt/vol), it was immediately transferred toan anaerobic chamber (Type A—manual air lock; Coy Inc., Grass Lake,Mich., USA) containing an atmosphere of Formier gas (N₂:H₂, 95:5%,vol/vol). After cooling to room temperature, 10 ml aliquots weredispensed into Hungate tubes, sealed, removed from the anaerobic chamberand subsequently sterilized by autoclaving at 121° C. for 20 min.Aerobic cultivations were conducted in 100 ml Klett flasks containing BMmedium lacking the reducing agents cysteine-HCl and Na₂S.9H₂O.

Luria Bertani (LB) medium (Sambrook, J. et al., Molecular cloning: aLaboratory Manual, 2nd ed. Cold Spring Harbor, N.Y.: Cold Spring HarbourLaboratory (1989)) was used for the purification of CGP degradingbacteria and for maintenance of viable cultures.

For the isolation of strain P. alcaligenes DIP1 a modified mineralmedium of Dufresne, A. et al., Macromolecules 31:6426-6433 (1998), wasused (SM-medium); 1.0 g NH₄Cl, 5.0 g KH₂PO₄, 1 g MgSO₄.7H₂O (sterilizedand added separately) and 10 ml of trace elements stalk solution, per Itap water. The trace elements stock solution contained nitrilotriaceticacid (70 mM, pH 6.5), FeSO₄.7H₂O (5 g/l), MnCl₂.4H₂O (0.85 g/l),CoCl₂.6H₂O (0.14 g/l), CuCl₂.2H₂O (0.085 g/l), H₃BO₃ (0.17 g/l),ZnSO₄.7H₂O (0.9 g/l), and Na₂MoO4.2H₂O (0.09 g/l). pH of the medium wasadjusted to 7.0 and subsequently sterilized by autoclaving at 121° C.for 20 minutes.

Liquid cultivations were applied in Erlenmeyer flasks with baffles andincubated on a Pilotshake RC-4/6-W horizontal shaker (Kühner AG,Birsfelden, Switzerland) at 150 rpm. To prepare CGP overlay agar plates,1.2% (wt/vol) bacto-agar was added to a CGP suspension (1-2 g/l), thismixture was subsequently sterilized by autoclaving at 121° C. for 20minutes and after cooling to 45° C., it was poured as thin layers overthe previously prepared SM agar plates.

To prepare CGP-overlay agar plates, 1.2% (wt/vol) bacto-agar was addedto a CGP suspension (1-2 g l⁻¹), this mixture was subsequentlysterilized by autoclaving at 121° C. for 20 minutes and after cooling to45° C., it was poured as thin layers over previously prepared SM agarplates.

Source and isolation of CGP: “Recombinant” CGP was isolated fromlyophilized cells of Ralstonia eutropha H16-PHB⁻4-Δeda(pBBR1MCS-2::cphA₆₃₀₈/edaH16) (Voss, I. et al., Metabol. Eng. 8:66-78(2006)) and purified according to a modified acid extraction method(Frey, K. M. et al., Appl. Environ. Microbiol. 68:3377-3384 (2002)). CGPwas isolated from Spirulina commercial products according to the methoddescribed previously for cyanobacteria (Simon, R. D. et al., Biochim.Biophys. Acta 420:165-176 (1976)).

To isolate and purify CGP on larger scales, the acid extraction method(Frey, K. M. et al., Appl. Environ. Microbiol. 68:3377-3384 (2002)) wasoptimized as follows; The CGP-containing dry mass was suspended in tapwater to give a final concentration of 0.1 g/ml. pH was reduced to 1with concentrated HCl (32%) and stirred overnight. The suspension wascentrifuged at 17000 rpm with CEPA Z61 continuous centrifuge (CEPA, CarlPadberg Zentrifugenbau GmbH, Lahr, Germany), the pellet was re-suspendedin 20 l HCl 0.1 N, stirred for 1 h, centrifuged again and thesupernatant was added to the first charge while the pellet wasdiscarded. The CGP-containing supernatant was neutralized (pH 7.3) withNaOH (50%) in 30 l glass bottles so that CGP precipitates. The milkysuspension was left to settle overnight in 4° C. before the supernatantwas discarded. CGP was repeatedly solved and neutralized for 3 moretimes to remove all impurities, which are insoluble in diluted HCL. Theresulting CGP was resolved in 30 l of HCL 0.1 N and passed through a 0.2μm Sartobran-P filter unit type 00 (Sartorius AG, Göttingen, Germany).The solution was neutralized again (pH 7.3) with NaOH, left overnight tosettle, the supernatant was discarded. To remove any water-solubleimpurities and desalt CGP, the pellet was washed 3 successive times with5 bed volumes of distilled water. Finally, CGP pellet was centrifuged at20000 rpm (CEPA Z41 contentious centrifuge), frozen at −30° C. andlyophilized in a BETA 1-16 type freeze-dryer (ChristGefriertrocknungsanlagen, Osterode, Germany).

Sterilization of CGP: To prepare sterile stock suspensions of CGP,diethyl ether was added to CGP with ratio 3:1 (vol/wt). After 15 min,the solvent was discarded and after complete evaporation a finesuspension was obtained by dissolving CGP in sterile 0.1 N HCl and thenprecipitating the polymer at pH 7.3 by adding an equal volume of sterile0.1 N NaOH. Alternatively, CGP was first dissolved in 0.1 N HCl, passedthrough a filter (pore size 0.2 μm, Millipore GmbH, Eschborn, Germany)and finally re-precipitated as described above. The concentrations ofCGP in stock solutions were adjusted by sedimentation of CGP upon shortcentrifugation (3500×g for 2 minutes) or by simply leaving CGP tosediment overnight at 4° C. In both cases, the supernatant was thenpartially discarded to finally obtain the desired concentration of CGP.Experiments for anaerobic CGP degradation were conducted inanaerobically prepared Hungate tubes containing 10 ml BM with or without0.5 g l⁻¹ yeast extract. Sterile CGP suspensions were injected directlyinto the anaerobically prepared BM Hungate tubes to final concentrationsof 1 g l⁻¹. The visual disappearance of CGP in the tubes indicated itsdegradation. To prepare CGP overlay agar plates, 1.2% (wt/vol)bacto-agar was added to a CGP suspension, this mixture was subsequentlysterilized by autoclaving at 121° C. for 20 min and after cooling to 45°C. it was poured as thin layers over the previously prepared BM agarplates.

Anaerobic degradation of CGP by samples of mammalian, avian and fish gutflora: To simulate the natural conditions of the habitats where theflora samples were obtained from, the prepared samples were used asinoculum and as nutritional supplement at the same time. Sterileanaerobic Hungate tubes containing 1 g l⁻¹ CGP in BM medium (withoutyeast extract) were inoculated to a final concentration of 10% andincubated at 37° C. until CGP degradation occurred.

Screening for CGP degrading bacteria in gut flora: To isolate CGPdegrading bacteria, 100 μl aliquots of the prepared flora samples werespread on aerobically prepared BM agar plates with CGP-overlays. Duringincubation for several days at 37° C., plates were inspected for theappearance of halos caused by CGP degrading bacterial colonies. Alsoduring incubation, a colony count procedure was applied to determine theratios between CGP degrading bacterial colonies and the non-degradingones.

Isolation of CGP degrading axenic cultures: Morphologically distinct CGPdegrading bacterial colonies were selected and further purified bytransferring material from colonies showing degradation halos to freshCGP-overlay agar plates for three successive generations. Further threepurification steps were applied on LB agar plates before the axenicculture were tested on CGP-overlay agar plates to confirm that theyretained the capability to degrade CGP.

Purity control: The purity of the isolated axenic cultures wasperiodically confirmed by microscopy and also by cultivation ondifferent media under anaerobic as well as aerobic conditions.

Analytical techniques: Free amino acids and small peptides(CGP-dipeptides) were detected by reversed phase HPLC (KontronInstruments, Neufahrn, Germany) after pre-column derivatization of theirfree amino groups with o-phtaldialdehyde (OPA) reagent as describedbefore (Aboulmagd, E. et al., Arch. Microbiol. 174:297-306 (2000)). Foranalysis of CGP samples, the polymer was in advance subjected to acidhydrolysis (6 N HCl, 95° C., overnight). Similarly, acid hydrolysis wasapplied for the qualitative and the quantitative confirmation of thedipeptide-constituting amino acids. The HPLC system was equipped with aB801 column (Prep Nova-Pak HR 3.9×300) (Knauer GmbH, Berlin, Germany),and equilibrated with starting eluent (81%, vol/vol, Na-acetate (50 mM):19%, vol/vol, methanol). OPA derivatives of amino acids were eluted witha methanol gradient (19-75%, vol/vol) at 40° C. and with flow rate of1.0 ml/min, and then detected fluorometrically at 330/450 nm(excitation/emission) employing a model 1046A fluorescence detector(Hewlett Packard, Waldbronn, Germany). For calibration,chromatographically pure amino acids were used (Kollection AS-10 fromServa Feinbiochemica, Heidelberg, Germany) or using dipeptides producedby total enzymatic hydrolysis of CGP employing the extracellular CGPaseof P. alcaligenes DIP1 (CphE_(al)) (Sallam, A. et al., Submitted forpublication (2008)).

Bacterial growth was monitored by measuring the increase in turbidity at578 nm after insertion of the Klett-flasks into an Eppendorf 1101Mspectrophotometer (Eppendorf, Hamburg, Germany).

Thin layer chromatography (TLC) analysis was applied on Silica gel 60plates (Merck, Darmstadt, Germany), the starting eluent of the HPLC wasused as rum buffer also for TLC, for staining, 20% (wt/vol) Ninhydrinsolution in aceton was applied. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) was performed in 11.5% (wt/vol) gelsafter Laemmli, U. K., Nature 227:680-685 (1970). Proteins andcyanophycin were stained with Coomassie staining method (Weber, K. etal., J. Biol. Chem. 244:4406-4412 (1969)) and afterwards withsilver-staining method (Heukeshoven, J. et al., Electrophoresis6:103-112 (1985)) to render proteins with lower concentrations visible.

DNA extraction and analysis of 16S rRNA genes: The isolation of totalgenomic DNA from axenic cultures was performed as described before (Rao,R. N. et al., Methods Enzymol. 153:166-198 (1987)). 16S rRNA genes wereamplified by PCR from total DNA using standard oligonucleotide primers(MWG-BIOTECH AG, Ebersberg, Germany). PCR products were purified using aNucleo-trap CR kit (Macherey-Nagel, Düren, Germany) and then directlysequenced. DNA sequencing was performed in custom at the institute forclinical chemistry and laboratory medicine (W.W.U. Münster, Germany) ona capillary sequencer (ABI Prism 3730 DNA analyser) and sequences wereanalyzed by data collection software v3.0 (both from Applied Biosystems,Darmstadt, Germany). Sequence reactions were prepared using BigDye®terminator v3.1 cycle sequencing kit (Applied Biosystems, Darmstadt,Germany) according to procedures indicated by the manufacturer, and thefollowing sequencing primers:

27f (5′-GAGTTTGATCCTGGCTCAG-3′; SEQ ID NO: 1), 343r(5′-CTGCTGCCTCCCGTA-3′; SEQ ID NO: 2), 357f(5′-TACGGGAGGCAGCAG-3′; SEQ ID NO: 3), 519r(5′-G(T/A)-ATTACCGCGGC(T/G)GCTG-3′; SEQ ID NO: 4), 536f(5′-CAGC(C/A)GCCGCGGTAAT(T/A)C-3′; SEQ ID NO: 5), 803f(5′-ATTAGATACCCTGGTAG-3′; SEQ ID NO: 6), 907r(5′-CCGTCAATTCATTTGAGTTT-3′; SEQ ID NO: 7), 1114f(5′-GCAACGAGCGCAACCC-3′; SEQ ID NO: 8), 1385r(5′-CGGTGTGT(A/G)CAAGGCCC-3′; SEQ ID NO: 9) and 1525r(5′-AGAAAGGAGGTGATCCAGCC-3′; SEQ ID NO: 10)

(MWG-BIOTECH AG, Ebersberg, Germany).

Sequence analysis and alignment, as well as the construction of thephylogenetic tree were applied as described previously (Sallam, A. etal., Submitted for publication (2008)): nucleic acid sequence data wereanalyzed with the Contig Assembly Program (CAP) online software.Sequences were aligned with previously published sequences ofrepresentative strains and other bacteria using the blast functionavailable on the National Center for Biotechnology Information (NCBI)database. Reference sequences were aligned using the ClustalX 1.8software and the phylogenetic tree was constructed using the programsTreeView 1.6.5 and NJplot. Bootstraping was applied to evaluate the treetopology by performing 100 resemblings.

Cultivation at 500 l scale: Cultivation at 500 l scale was performed ina Biostat D650 stainless steel bioreactor (B. Braun BiotechInternational, Melsungen, Germany), which had a total volume of 650 l(64 cm inner diameter and 198 cm height) and a d/D value relation(relation of stirrer diameter to vessel diameter) of 0.375. Thisbioreactor was equipped with three stirrers, each containing six paddlesand a Funda-Foam mechanical foam destroyer (B. Braun BiotechInternational, Melsungen, Germany). In addition, ports were used forsterilizable probes to measure dissolved oxygen (pO₂) (model 25; MettlerToledo GmbH, Steinbach, Switzerland), pH (model Pa/25; Mettler-ToledoGmbH), foam (model L300/Rd. 28; B. Braun Biotech International,Melsungen, Germany), temperature (pt 100 electrode; M. K. Juchheim GmbH,Fulda, Germany), and optical density at 850 nm (model CT6;Sentex/Monitek Technology Inc.). The operations were controlled andrecorded by a digital control unit in combination with the MFCS/winsoftware package (B. Braun Biotech International). Cultivations weredone at 30° C. and pO₂ was adjusted to exceed 40% saturation in themedium, which was automatically controlled by stirring, aeration ratewas kept stable at 0.7 vvm (volume per volume×minute). The initial pH ofthe medium was 6.9 and its increase up to 7.5 during growth wastolerated, otherwise, pH was controlled by the addition of 4 N HCl.

Cell separation, concentration, and desalting of supernatant-proteins:Cells were harvested by centrifugation with a CEPA type Z41 or type Z61continuous centrifuges (Carl Padberg Zentrifugenbau GmbH, Lahr,Germany). Proteins with molecular size over 30 kDa were concentrated andsubsequently desalted (5 bed volumes of H₂O) using a cross flowSartocon® polyethersulfone-cassette with a COP (Cut-Off Point) of 30 kDaand a stainless steel holder of type Sartocon® 2 Plus (Sartorius AG,Göttingen, Germany).

Growth on different substrates: Substrate utilization was investigatedin 100 ml Klett-flasks with baffles; each flask contained 10 ml of SMmedium and 1 g l⁻¹ of one of the following substrates: lactate, citrate,succinate, acetate, propionate, gluconate, glucose, fructose, sucrose,and glycerine. Stock solutions of all substrates were sterilized byfiltration (pore size 0.2 μm, Millipore GmbH, Eschborn, Germany).Experiments were performed in duplicates, which were inoculated from apreculture grown under the same test conditions. Growth was monitored bythe increase in OD_(578 nm) after an incubation period of 24 h at 30° C.

Fermentative production of cyanophycin: CGP was produced in 500-l scalefermentation using a recombinant strain of E. coli DH1 harboring plasmidpMa/c5-914::cphA_(PCC6803) (Frey, K. M. et al., Appl. Environ.Microbiol. 68:3377-3384 (2002)). As substrate and medium, the previouslyapplied protamylasse (Avebe, Veendam, The Netherlands) medium was usedand prepared by dilution, sieving and centrifugation as described by(Elbahloul, Y. et al., Appl. Environ. Microbiol. 71:7759-7767 (2005)).

To determine the optimum concentration of the available charge ofprotamylasse for CGP production, 100 ml cultivations on protamylassemedium with concentrations between 4-8% (vol/vol) were tested in 200 mlbaffled flasks; the medium had pH of 7.5 and contained 100 mg l⁻¹ampicillin. After inoculation, flasks were incubated at 37° C. for 15 hunder shaking (120 rpm) (shaker type G 25, New Brunswick ScientificGmbH, Nürtingen, Germany). Finally, CGP content was estimated in a 50 mlsample from each flask.

Prior to fermentation, 16-l of preculture was cultivated in 2-lbaffled-flasks each containing 1-l of protamylasse medium (7%; vol/vol,pH 7.5), 100 mg l⁻¹ ampicillin and 0.15% (vol/vol) silicon-antifoamemulsion (50%; vol/vol, Wacker Silicones, Burghausen, Germany) and wereincubated at 30° C. for 20 h under shaking (110 rpm, shaker type RC-6-W,Adolf Kühner AG, Birsfelden, Switzerland).

Main cultivation was performed in a Biostat D650 stainless steelbioreactor (B. Braun Biotech International, Melsungen, Germany) withtotal volume of 650 l (64 cm inner diameter and 198 cm height) and a d/Dvalue (stirrer diameter to vessel diameter) of 0.375. The bioreactor wasequipped with three stirrers, each containing six paddles and aFunda-Foam mechanical foam destroyer (B. Braun Biotech International,Melsungen, Germany). In addition, ports were used for sterilizableprobes to measure dissolved oxygen (pO₂) (model 25; Mettler Toledo GmbH,Steinbach, Switzerland), pH (model Pa/25; Mettler-Toledo GmbH), foam(model L300/Rd. 28; B. Braun Biotech International, Melsungen, Germany),temperature (pt 100 electrode; M. K. Juchheim GmbH, Fulda, Germany), andoptical density of 850 nm (model CT6; Sentex/Monitek Technology Inc.).Operations were controlled and recorded by a digital control unit incombination with the MFCS/win software package (B. Braun BiotechInternational).

The bioreactor was filled with 400-l of 7% protamylasse-medium (pH 7.5)and 75 ml antifoam solution, in-situ sterilized and after cooling to 30°C., it was inoculated with 4% (vol/vol) of preculture after addition of100 mg l⁻¹ sterile ampicillin solution. Cultivation was run at 30° C.for the first 6 h, pH of the medium was maintained at 7.5 by theaddition of 6 M NaOH or 6 M HCl. pO₂ was kept constant at 20% and wasadjusted automatically by stirring, aeration rate was kept stable at0.17 vvm (volume per volume×minute). Foaming was controlledautomatically by the mechanical foam-destroyer or by the addition ofantifoam sterile solution (50%, vol/vol). After 6 h of incubation,temperature was elevated to 37° C. until the fermentation wasterminated. Cells were harvested by centrifugation with a CEPA type Z61continuous centrifuge (Carl Padberg Zentrifugenbau GmbH, Lahr, Germany).

Large-scale extraction and purification of CGP: To obtain pure CGP fromthe resulting wet biomass, the previously optimized acid extractionmethod for large-scale extraction and purification of CGP was applied(Sallam, A., and A. Steinbüchel. 2008b. Biotechnological process for thetechnical production of β-dipeptides from cyanophycin. Underpreparation). Finally, the extracted CGP was frozen at −30° C. andlyophilized in a BETA 1-16 type freeze-dryer (ChristGefriertrocknungsanlagen, Osterode, Germany).

Sterilization of CGP: CGP was sterilized by diethyl ether or by solvingin 0.1 N HCl and sterile-filtration as described before (Sallam, A., andA. Steinbüchel. 2007b. Anaerobic and aerobic degradation of cyanophycinby the denitrifying bacterium Pseudomonas alcaligenes strain DIP1-Roleof other three co-isolates in the mixed bacterial consortium. Submittedfor publishing). The desired concentrations of CGP in stock solutionswere adjusted by sedimentation of CGP upon short centrifugation (2800×gfor 2 min) or by leaving CGP to settle overnight at 4° C., thesupernatant was then partially discarded to obtain the desiredconcentration of CGP.

Analytical techniques: Bacterial growth and CGP degradation weremonitored by measuring the change in turbidity after insertion ofKlett-flasks into a Klett-photometer (Manostat Corporation, New York,USA), or by measuring OD in 1 ml samples at 600 nm with a Libra S5photometer (Biochrom Ltd., Camebridge, UK). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in11.5% (wt/vol) gels after Laemmli, U. K., Nature 227:680-685 (1970).In-gel-renaturation of proteins was applied after (Lacks, S. A. et al.,J. Biol. Chem. 225:7467-7473 (1980)). Proteins and cyanophycin werestained with Coomassie staining method (Weber, K. et al., J. Biol. Chem.244:4406-4412 (1969)) or with silver-staining method (only for proteins)(Nesterenko, M. V. et al., J. Biochem. Biophys. Methods 3:239-242(1994)). Concentrations of total protein and CGP were determined usingBradford reagent as described by (Elbahloul, Y. et al., Appl. Environ.Microbiol. 71:7759-7767 (2005)). large-size proteins in cultivationsamples were concentrated, desalted and separated from CGP-dipeptides byultrafiltration using 10 kDa-membrane Vivaspin tubes (Vivascience AG,Hannover, Germany), or using an Amicon-chamber (Amicon, Beverly, USA)with 10 kDa-membranes (Millipore Corporation, Bedford, USA) for largervolumes. Free amino acids and dipeptides were detected by reversed phaseHPLC (Kontron Instruments, Neufahrn, Germany) after pre-columnderivatization of their free amino groups with Orthophtaldialdehyde(OPA) reagent as described before (Aboulmagd, E. et al., Arch.Microbiol. 174:297-306 (2000)). For analysis of constituting amino acidsof CGP or CGP-dipeptide, samples were subjected in advance to acidhydrolysis (6 N HCl, 95° C., overnight). HPLC system was equipped with aB801 column (Prep Nova-Pak HR 3.9×300) (Knauer GmbH, Berlin, Germany)and equilibrated with starting eluent (81%, vol/vol, Na-acetate (50 mM):19%, vol/vol, methanol). OPA derivatives of amino acids were eluted by amethanol gradient (19-75%, vol/vol) at 40° C. with 1.0 ml min⁻¹ flowrate, and then detected fluorometrically at 330/450 nm(excitation/emission) employing a model 1046A fluorescence detector(Hewlett Packard, Waldbronn, Germany).

Determination of CphE_(al) activity and concentration: The activity ofthe cyanophycinase (CphE_(al)) was inspected by formation of degradationhalos on CGP-overlay agar plates by small aliquots of concentratedenzyme solutions, for this, 5 ml culture samples were centrifuged at2800×g for 30 min (Megafuge 1.0 R, Heraeus Sepatech GmbH, Osterode,Germany) and 4 ml of the resulting supernatant were concentrated 100folds by ultrafiltration. For the quantitative determination of theenzyme in culture samples, a photometric method was developed asfollows; 2 μl of the concentrated culture supernatant were added to 1 mlof a CGP suspension (100 mg l⁻¹) and incubated at 30° C. for 30 min in atube rotator (3 rpm, type 502 S, Watson-Marlow GmbH, Rommerskirchen,Germany). Finally, OD_(600 nm) of samples was measured to indicate thedecrease in CGP amount due to degradation; this in turn was used toindicate the concentration of CphE_(al) in solutions through therespective calibration curve.

Optimal concentrations and conditions for CGP degradation: Using a crudeCphE_(al) powder obtained during a previous 500-l fermentation with P.alcaligenes strain DIP1 (Sallam, A., and A. Steinbüchel. 2008b.Biotechnological process for the technical production of β-dipeptidesfrom cyanophycin. Under preparation), and to determine the optimum ratiobetween CGP concentration and crude CphE_(al) in relation to therequired degradation time, serial dilution (50-100 g l⁻¹) of pure CGPsuspensions (in water, pH 7.3) were prepared in Eppendorf tubes withtotal volume of 1 ml each, different amounts of crude CphE_(al) powder[4.6% (wt/wt) CphE_(al) content] were added to each prepared CGPconcentration, reaction tubes were incubated at 30° C. in a tube rotator(3 rpm) to reveal the required incubation periods for complete CGPdegradation.

Experiments to determine the optimum pH for CGP degradation wereconducted in Eppendorf tubes containing 1 ml CGP suspension (100 g l⁻¹)with pH values between 5.0 and 9.0, reaction tubes contained in addition10 g l⁻¹ crude CphE_(al) and were incubated at 30° C. for 30 min.Reaction mixtures for optimum degradation temperature contained similarconcentrations of CGP (pH 7.0) and CphE_(al) and were incubated at 15,20, 25, 30, 35, 37, 40, 50, 60 or 70° C. for 30 min. After bothexperiments, CGP degradation in all tubes was calculated in percent andcompared together.

Purification of CphE_(al) by organic solvents and ammonium sulfateprecipitation: 1 ml of concentrations between 10-100% (vol/vol) of coldethanol, acetone or methanol were added to 50 μl of a concentrated crudeCphE_(al) solution (14 g l⁻¹) in Eppendorf tubes, reactions mixtureswere incubated for 60 min at −20° C. After reaction tubes werecentrifuged for 5 min at 16000×g, the pellets were dried andre-suspended in 50 μl sodium phosphate buffer (pH 7.0). Ammonium sulfatefractionation was applied by the stepwise increase in thepercent-ammonium sulfate saturation (10-100%) in 10 ml of crudeCphE_(al) solution (3.5 g l⁻¹), in each step, tubes were incubated atroom temperature for 30 min and centrifuged for 10 min at 16000×g,pellets were suspended in sodium phosphate buffer (pH 7.0) then desaltedby ultrafiltration. All protein pellets were assayed for CGP degradationon CGP-overlay agar plates as well as for protein content in SDS-PAGE.

Specific substrate purification of CphE_(al): A purification method forCphE_(al) was developed depending on the strong affinity of the enzymein the crude extract to its insoluble substrate (CGP), where onlyCphE_(al) binds to CGP and thus can be harvested by precipitation. Todetermine the time required for the complete binding of the enzyme toCGP at pH 7.0, 0.5 ml of a concentrated crude CphE_(al) solution (14 gl⁻¹) was added to 0.5 ml of a CGP suspension (100 g l⁻¹). After each ofthe first 10 incubation minutes, 2 μl aliquot from the reactionsupernatant (after short centrifugation) was tested for degradationactivity on CGP-overlay agar plates, diminishing of degradation halosindicated complete binding of CphE_(al) to CGP. The actual purificationprocesses was performed in a similar 1 ml reaction mixture and proceedas follows: after the complete binding of CphE_(al) to CGP, the mixturewas centrifuged for 30 seconds (16000×g), supernatant was discarded andthe pellet was washed 5 times with 10 ml of sodium phosphate buffer (50mM). Afterwards, the pellet was suspended in 1 ml phosphate buffer andincubated overnight at 30° C. under rotation (3 rpm) until the completedegradation of CGP occurred. The mixture was centrifuged (5 min,16000×g), CGP-dipeptides were then removed by ultrafiltration and theconcentrated protein fraction of the supernatant was analyzed for purityby SDS-PAGE.

Characterization of CphE_(al) from P. alcaligenes strain DIP1: Todetermine temperature stability of the purified CphE_(al), 10 μlaliquots were incubated for 20 min at different temperatures (10-80°C.), 3 μl thereof were then tested for degradation activity onCGP-overlay agar plates at 30° C. For the optimum degradationtemperature, 3 μl aliquots of the purified CphE_(al) solution were addedto 1 ml CGP suspensions (100 g l⁻¹ in 50 mM Na-phosphate buffer, pH 7.0)and incubated for 20 min at different temperatures (10, 20, 30, 40, 45,50 and 60° C.). For the optimum degradation pH of CGP by the purifiedCphE_(al), 3 μl aliquots of the purified CphE_(al) stalk solution wereadded to 1 ml CGP suspensions (100 g l⁻¹ in 50 mM Na-phosphate buffer)with different pH values (5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 or 9) andincubated for 30 min at 30° C. Finally, CGP degradation was determinedphotometrically in reaction tubes of both experiments as describedabove. Substrate specificity of purified CphEal was tested as describedbefore (Obst, M. et al., Biomacromolecules 5:153-161 (2004)) on thefollowing polypeptide substrates CGP, bovine casein (Hammersten-grade)(Merck, Darmstadt, Germany), bovine serum albumin (BSA) (Roth,Karlsruhe, Germany), and poly(α,β-D/L-aspartic acid) (Bayer AG,Leverkusen, Germany).

To reveal the effect of enzyme inhibitors on the purified CphE_(al), 50μl aliquots of the purified enzyme stalk solution were added to 450 μlof Na-phosphate buffer (50 mM) and incubated for 2 h at 30° C. in thepresence of one of the following Group-specific inhibitors; Leupeptin(thiol-proteases), EDTA (metalloproteases), Pefabloc (serine-proteases),PMSF (serine-proteases) or N-bromosuccinimide (tryptophan residues). 5μl sample of each reaction mixture was assayed for activity onCGP-overlay agar plates. Afterwards, 5 μl aliquots of a CGP stalksuspension (50 g l⁻¹) were added to the reaction tubes, incubated forfurther 15 min, centrifuged, and screened for degradation products viaHPLC.

Example 1 Anaerobic degradation of CGP by samples from mammalian, avianand fish gut flora: The filtered flora samples contained a large numberof different bacteria as well as high content of substrates. To simulatethe natural conditions inside the digestive tracts where these sampleswere obtained from, the flora solutions were used as inoculum and asnutritional supplement at the same time. Sterile anaerobic Hungate tubescontaining BM medium and 1 g l⁻¹ CGP as sole substrate were inoculatedin a concentration of 10%. All inoculated anaerobic tubes showedcomplete CGP degradation under these conditions. The incubation periodsrequired for the complete degradation of CGP in the tubes ranged from 12to 48 h.

Screening for CGP degrading bacteria in gut flora: To isolate CGPdegrading bacteria, 100 μl aliquots of the prepared flora samples werespread on CGP-overlay agar plates. During incubation for several days at37° C., plates showed that occurrence of CGP degrading bacteria variedlargely among different flora (Table 1). The highest incidence was incase of cecum flora from rabbit (Egypt), sheep (Germany) and digestivetract flora from carp fish (Germany). Also the morphologicalcharacteristics of the degrading colonies, the required incubationperiod, form and intensity of halos caused by CGP degradation variedlargely among different flora samples as well as among colonies withineach sample. This indicated the presence of distinctive bacterialspecies with diverse capabilities of CGP degradation.

Isolation and purification of CGP degrading strains: Morphologicallydistinct CGP degrading bacterial colonies were selected and furtherpurified by transferring colonies showing degradation halos to freshCGP-overlay agar plates. During purification work, many CGP degradingcolonies lost their ability to induce degradation halos as axeniccultures and were therefore disregarded. Similarly, several CGPdegrading consortia showed extreme difficulties to be obtained as axeniccultures and were also omitted from further purification procedures. Onthe other hand, the purification phase resulted in 62 axenic cultures,which showed morphologically distinctive characteristics with retainedcapability of CGP degradation. As demonstrated in Table 1, CGP degradingbacteria were isolated from different sites along the digestive tract ofeach animal. This is in accordance with the facts known about thedegradation of dietary protein for example in ruminants; while the rumenis the major degradation site for dietary protein, undigested protein(bypass or escape protein) moves with the digesta into the lowerdigestive tract, where a portion thereof is broken down by the animal'senzymes or by the lower gut flora to be then absorbed before the restgets excreted. The degradation of bypass protein is important forspecific physiological functions such as milk production (Holter, J. B.et al., J. Dairy. Sci. 76:1342-1352 (1993)).

Degradation of CGP by axenic cultures under anaerobic and aerobicconditions: The 62 axenic cultures were examined for the capability todegrade CGP in liquid media under aerobic conditions. This was appliedin BM medium containing 1 g l⁻¹ CGP and 0.5 g l⁻¹ yeast extract andsignified the capability of all axenic cultures to degrade CGP (Table1). Anaerobic CGP degradation experiments in Hungate tubes containingsimilar medium in addition to reducing agents, revealed that from the 62isolated strains, 46 could degrade CGP anaerobically as well. Of those,38 degraded CGP completely while the rest showed partial CGPdegradation. The incubation periods required for the anaerobic CGPdegradation by axenic cultures ranged from 24 h to 7 days at 37° C.

Degradation products of CGP: Directly after the complete degradation ofCGP by the 62 pure isolates in aerobic and anaerobic cultures,degradation products of CGP in culture supernatants were determined byHPLC after pre-column derivatization with OPA reagent as mentioned inthe methods section. As expected, HPLC analysis revealed that allisolates degrade CGP to its constituting dipeptides. Similar analysisfor supernatant samples that have been subjected in prior to acidhydrolysis revealed the presence of the three CGP-constituting aminoacids; aspartate, arginine and lysine. The detection of the latter threeamino acids is in accordance with the known composition of recombinantCGP which was applied for these experiments, where approximately 6 Mol %of the arginine moieties are replaced with lysine (Voss, I. et al.,Metabol. Eng. 8:66-78 (2006)).

Taxonomic affiliation of the CGP degrading axenic cultures: Themorphological characteristics of the isolated axenic cultures suggestedthat approximately 50% of the isolates belong to the genera Bacillus andPseudomonas, while the remaining isolates belong to other generaincluding Streptomyces and Micromonospora. To provide a clearer insighton the phylogeny of the CGP degrading bacteria in the examined gutflora, eight strains from the 62 were further identified by 16S rDNAsequencing. As shown in table 1, three isolates; 17, 35, 49 from rumenflora of sheep (Germany), cecum flora of turkey (Egypt), and thedigestive tract flora of tilapia fish (Egypt), respectively, wereidentified as members of the genus Bacillus with 16S rDNA sequencesimilarity of over 99% to B. megaterium AC46b1, B. megaterium MPF-906and B. Koguryoae SMC4352-2^(T), respectively. Two isolates; 15 and 47from colon flora of cow (Germany) and cecum flora of pigeon (Egypt) werefound to belong to the genus Brevibacillus with 16S rDNA sequencesimilarity of over 98% to B. reuszeri DSM9887^(T) and Brevibacillus sp.SE004, respectively. Strain 31, isolated from rabbit cecum (Germany) wasaffiliated to the genus Streptomyces with sequence similarity of over98% to S. avermitilis NCIMB 12804^(T). 16S rDNA sequence analysis alsoshowed that strain 38 from duck cecum (Germany) belongs to genusMicromonospora and has a sequence similarity of over 99% toMicromonospora sp. 0138. Finally, a representative of genus Pseudomonaswas also found among the identified isolates; strain 52 from carp fish(Germany) showed 16S rDNA sequence similarity of over 99% to the CGPdegrading bacterium P. alcaligenes DIP1.

The constructed phylogenetic tree (FIG. 1) shows the relationshipbetween the eight identified strains from diverse animal, avian and fishflora to their closely related strains and to other bacteria. Moreover,the taxonomic position of all known bacteria capable of extracellularCGP degradation is demonstrated in the phylogenetic tree. Further Table2 provides an overview on the wide spread, habitats, and phylogeny ofbacteria capable of extracellular CGP degradation.

CGP in Spirulina platensis commercial products: The market nameSpirulina, which is often associated with several nutritional benefitsand thus used as additives for human and animals (Narasimha, D. L. R. etal., J. Sci. Food Agric. 33:456-460 (1982); Kihlberg, R., A. Rev.Microbiol. 26:427-466 (1972); Lu, J. et al., Aquaculture 254:686-692(2004); Ross, E., Poult-Sci. 69:794-800 (1990))). To investigate whetherthis is associated with the presence of CGP, three Spirulina productsavailable on the German market were inspected for the presence of CGP.Two products; from Sanatur GmbH (Singen, Germany) and Greenvally GmbH(Berlin, Germany) consisted of 100% Spirulina platensis, while the thirdvariant from Aurica GmbH (Schwalbach-Elm, Germany) contained 40%thereof. 10 g CDM of each of the three products that correspondsapproximately the double of the usual daily dose were prepared bygrinding to a fine powder before CGP isolation. The three marketvariants showed CGP contents of 0.06%, 0.13% and 0.15%, respectively.Confirmatory HPLC analysis of hydrolyzed samples of the isolated CGPrevealed, as expected, the typical constituting amino acids ofcyanobacterial CGP; aspartate and arginine.

Example 2: Development of an optimal process for the production ofβ-dipeptides from CGP: Several factors were first optimized on smallscale, for example; a required suitable and economic media, highlyconcentrated suspensions of CGP which can be practically prepared fordegradation, and to define the necessary amount of the extracellularenzyme to achieve the complete CGP-degradation in a relatively shortincubation period.

The change in OD_(600 nm) During the production of CphE due to theaddition of the inductor (CGP) and its subsequent degradation,represents a definite sign for the release of the CphE, therefore, aclear medium and a controlled growth of cells were necessary, this wasachieved through the final medium composition mentioned in materials andmethods section. Moreover, among the tested substrates, strain P.alcaligenes DIP1 showed best growth on citrate (FIG. 3), the appliedconcentration of citrate (1 g/l) as sole substrate guaranteed for areasonable but in the same time controlled growth turbidity of culturesduring the induction and degradation phases.

To determine a balanced ratio between CGP and the crude CphEconcentrations for the degradation phase of the large scale process(phase III, see under), the following small scale experiments wereconducted; serial dilution (10-50 g/l) of pure CGP suspensions (inwater, pH 7.3) were prepared in Eppendorf tubes with total volume of 1ml each, Furthermore, a serial dilution (1-10 g/l) of crude CphE wastested on each prepared CGP concentration, the reaction tubes wereincubated at 30° C. in a tube rotator with rotation rate of 3 rpm. Theexperiments showed a relatively constant increase in CGP degradationtime when lower concentrations of crude CphE were used. The highesttested concentration of CGP (50 g/l) could be degraded within 10 h inthe presence of 2 g/l crude CphE powder (FIG. 4).

Large scale production of CGP-dipeptides: To enable the routineproduction of large quantities of pure CGP-dipeptides, a triphasicprocess was constructed; Phase I: large scale isolation and purificationof CGP, Phase II: large-scale production of crude CphE powder, PhaseIII: degradation of CGP to its dipeptides.

Phase I (large scale isolation and purification of CGP): To obtain alarge amount of CGP, 4776 g cell dry mass of CGP-containing cells wereused, this amount was previously produced during one 500 l fermentationwith Ralston/a eutropha H16-PHB⁻4-Δeda (pBBR1MCS-2::cphA₆₃₀₈/edaH16).CGP content of this biomass charge was 32% (wt/wt) (Voss, I. et al.,Metabol. Eng. 8:66-78 (2006)). Additionally, several other dry biomasscharges from lab-scale fermentations with R. eutrophaH16-PHB⁻4-Δldh/Ωkm-cphA₆₃₀₈ were mixed (a total of 2490 g) andconsidered as a secondary charge. CGP was extracted and purified fromeach charge separately according to the modified acid extraction methodmentioned in materials and methods section. The resulting CGP wet masswas 1948 g for the main cell charge and 295 g for the secondary charge,after dry-freezing of the purified CGP, 621.83 g and 82.17 g of pure CGPwere obtained from both charges, respectively.

HPLC analysis of the individual amino acid constituents of the isolatedCGP from both cell charges revealed that the polymer was mainly composedof aspartate and arginine and that it contained only little lysine of4.5% (mol/mol). SDS polyacrylamide gel electrophoresis revealed that theisolated CGP in both charges has a molecular weight of about 20 to 30kDa. Moreover, Both HPLC analysis and SDS polyacrylamide gelelectrophoresis indicated the lack of any impurities in the obtainedCGP. Thus, both CGP charges could be mixed together to a final amount of704 g of pure CGP.

Phase II (large scale production of crude CphE powder): To continuetowards the development of a process for the commercial production ofCGP-dipeptides, the production of enough amount of crude CphE from P.alcaligenes DIP1 was necessary, therefore, the strain was cultivated at500 l scale in a Biostat D650 bioreactor. The preculture of P.alcaligenes DIP1 was cultivated in 2 l baffled-flasks containing 1 l ofSM medium with 1 g/l sodium citrate; the flasks were incubated undershaking for 12 h at 30° C. The bioreactor was filled with 420 l of SMmedium containing 1 g/l sodium citrate, sterilized, and inoculated with4% (vol/vol) preculture. Initial pH was adjusted to 6.9. Duringfermentation, Cells of strain P. alcaligenes DIP1 started to growslightly after the first fermentation h and reached OD_(600 nm) value of0.4 to inter the stationary phase after nearly 9 h. The excretion ofCphE was induced by the addition of 0.25 g/l sterile CGP suspensionafter the first 2 h of the stationary phase, the addition of theinductor (insoluble CGP) increased the OD_(600 nm) immediately from 0.55to 0.98, the complete degradation of the added CGP required 2 h and wasindicated by the decrease of OD_(600 nm) to reach a value close to thatbefore induction. One h later, cells started to grow slightly which ismost probably due to the presence of CGP-dipeptides in the medium. Thefermentation was terminated after a total cultivation period of 14 h(FIG. 5).

For harvesting, concentration and desalting of proteins in thesupernatant including the excreted extracellular cyanophycinase, acontinuous system was prepared while the fermentation was running (FIG.6); for harvesting, a CEPA Z41 continuous centrifuge was used toseparate the cells while the supernatant was collected in a central 100l tank. To concentrate the collected supernatant simultaneously, a crossflow unit with a 30 kDa cassette was connected to the central tank; theconcentrated retentate containing proteins with molecular size largerthan the COP of the filter cassette (30 kDa) was re-pumped into thetank, while the permeate was directly discarded. The flow rate of thecross flow was adjusted to maintain the volume in the tank at about 50l. For cooling, an ice bag was introduced into the central tank tomaintain the temperature of the solution under 20° C. After harvestingcame to an end, the concentration process continued until only 5 lconcentrate remained in the tank. For desalting, the concentrate waswashed with 5 bed volumes of H₂O. During each of the previous steps,samples were collected and checked for CGP-degradation activity onCGP-overlay agar plates (FIG. 2). Finally, the concentrate was frozen at−30° C. and lyophilized to obtain a final dry weight of crude proteinpowder of 17.5 g.

Phase III (Degradation of pure CGP to its dipeptides): The large scaledegradation of CGP to its dipeptides was applied on 250 g pure CGP andusing 10 g of the crude CphE powder, these amounts represent thehighest—easy to prepare—CGP concentration (50 g/l) and the sufficientconcentration of crude CphE (2 g/l) to assure the complete degradationwithin 12 h as determined by the small scale experiments. The 250 g CGPpowder were resolved in 5 l of 0.1 M HCL, neutralized to pH 7.3, left tosediment in 4° C., and finally desalted by washing 3 times with tapwater to obtain the desired concentration in a total volume of 5 l.Afterwards, the 10 g of the crude CphE powder were added to the CGPsuspension and incubated for 12 h at 30° C. under weak stirring. Afterthe complete degradation of CGP, the dipeptide solution wassterile-filtered, separated from the crude protein components with thesame cross flow system used during phase II, frozen at −30° C., andfinally lyophilized to obtain 227.5 g of CGP-dipeptides.

In order to determine if further filtration steps are necessary for thepurification of CGP dipeptides, several 20 ml aliquots (taken before thelater freezing and lyophilization steps) were filtered with differentmembranes having the following COPs; 10, 5, 1, and 0.5 kDa. Afterfiltration, the loss of CGP-dipeptides was evaluated by determining thedry weight of lyophilized 1 ml aliquots before and after filtration, aswell as by HPLC analysis. Compared to the original dipeptide solution,the maximum dry weight loss during filtration was in the case of themembrane with the smallest COP (0.5 kDa) which was 78.5% (wt/wt), themembrane with 1 kDa caused a loss of 47.8% (wt/wt), while that with 5kDa caused a loss of 11.6% (wt/wt), the filter-membrane with higher COP(10 kDa) showed a minimal loss of 7.4% (wt/wt).

The purity grade of the dipeptides resulting from all filtration systemswas monitored by TLC as well as HPLC analysis, all filtered dipeptidesamples showed the same high grade of purity (FIG. 7, I). Thus, nofurther filtration steps were needed for the main dipeptide charge. Thecross flow with the 30 kDa cassette was clearly the most practicalinstrument also to regain the protein portion of the degraded CGPsolution. The regained protein solution was frozen and lyophilizedagain. The activity test for the regained powder on CGP-overlay agarplates showed that CphE survived all the previously mentioned proceduresand retained its activity (FIG. 2). The recovery rate for the crudepowder was 78%.

For the final quality control of the resulting dipeptide powder, directsamples as well as acid hydrolyzed samples were examined by TLC; onlyone spot was indicated for the direct samples (FIG. 7, I) while thehydrolyzed ones showed the typical spots for the standard amino acidsaspartate, arginine and lysine (FIG. 7, II). Also, the confirmatory HPLCanalysis for the acid hydrolyzed samples revealed only the typical peaksfor these 3 constitutive amino acids. On hand of these results, thepurity of the dipeptide powder was estimated to be over 99%. The finaloutcome (227.5 g pure dipeptides) from the degradation of 250 g CGPindicates that the overall efficacy of the whole process represents 91%,which may be even more increased through future optimizations.

Example 3. Fermentative production of cyanophycin at 500 l scale: Asufficient amount of CGP for this study was produced in 500-l scalefermentation after (Elbahloul, Y. et al., Appl. Environ. Microbiol.71:7759-7767 (2005)). However, the recombinant strain E. coli DH1(pMa/c5-914::cphA_(PCC6803)) was cultivated on 7% protamylasse(vol/vol); this concentration was proved to be optimum for CGPproduction during pre-experiments on the available charge ofprotamylasse.

During fermentation (FIG. 8) and after the first 6 h of incubation,temperature was elevated to 37° C. to enable the induction of theCGP-synthetase (CphA) gene (Frey, K. M. et al., Appl. Environ.Microbiol. 68:3377-3384 (2002)). Samples taken each h showed, asexpected, a progressive intracellular accumulation of the polymer whichreached maximum after 13 h (microscopically estimated) and remainedconstant afterwards (FIG. 9). OD_(600 nm) reached 18.3 after 15 h andthe fermentation was then terminated, however, later analysis revealedmaximum CGP accumulation of 13% (wt/wt of CDM) to be after 13 h offermentation and that it sank to 10% (wt/wt of CDM) at the harvest time(15 h). After CGP extraction from the resulting 4626 g wet-mass of cells(1372 g CDM), 135 g of pure CGP powder were obtained, HPLC analysis ofCGP indicated only the constitutive amino acids aspartate, arginine inaddition to lysine which represented 6.8 Mol % of the polymer. SDS-PAGEconfirmed the purity of CGP and showed a molecular weight of 25-30 kDa.

Optimum growth conditions for P. alcaligenes strain DIP1: TestingSM-medium with different concentrations of citrate (0.5-10 g l⁻¹), whichwas previously proved to be an optimal substrate for strain DIP1(Sallam, A., and A. Steinbüchel. 2008b. Biotechnological process for thetechnical production of β-dipeptides from cyanophycin. Underpreparation), showed that 6 g l⁻¹ produces maximal growth (475 Klettunits after 13 h at 30° C.). Cultures of strain DIP1 cultivated on 6 gcitrate under the same conditions but having different pH values(5.5-8.5) showed that strain DIP1 has an optimum growth pH of 6.5, whilecultivations at different incubation temperatures (15-45° C.) revealed37° C. as the optimum temperature for growth.

Optimum cultivation conditions for maximum CphE_(al) production by P.alcaligenes strain DIP1: To conclude the optimum cultivation conditionsfor maximum production of CphE_(al) by strain DIP1, cells werecultivated on different concentrations of citrate under optimum growthconditions, when cells reached the stationary phase; CphE_(al)production was induced by the addition of 0.25 g CGP. During thefollowing 5 h, supernatant samples were concentrated (100 folds) andscreened for degradation activity on CGP-overlay agar plates as well asphotometrically. Supernatant samples from cultures with citrateconcentrations below 0.5 or higher than 4 g l⁻¹ did not show anydegradation activity, while the maximum activity and subsequentlymaximum CphE_(al) production was monitored after 5 h of induction forcultures grown on 2 g l⁻¹ citrate. Further cultivation experiments underoptimal production conditions confirmed that cells of strain DIP1 reachthe stationary phase after about 13 h of incubation and proved this timepoint to be optimal for the induction of CphE_(al). Thus, the followingconditions were considered optimum for CphE_(al) production; SM-mediumwith 2 g l⁻¹ citric acid, pH 6.5, 37° C., induction after 13 h, andharvesting during the 16^(th) incubation h.

Optimum induction of CphE_(al) with CGP and alternative inductors:Besides CGP, other possible inductors were tested in cultures of strainDIP1; β-Dipeptide from CGP, synthetic dipeptides (α-arginine-aspartate,α-lysine-aspartate, α-ornithine-aspartate) (Sigma-Aldrich, St. Louis,Mo., USA), α-polyaspartate (Bayer AG, Leverkusen, Germany),poly-ε-Lysine (Chisso, Tokyo, Japan), L-aspartate, L-arginine, L-lysine,L-citrulline, L-ornithine as well as aspartate analogues[N-acetyl-aspartate, ureidosuccinic acid (N-Carbamoyl-aspartate)]. Allinductors were tested first at a concentration of 0.25 g l⁻¹ andCphE_(al) production was assayed on CGP-overlay agar plates and alsophotometrically. Only CGP, the dipeptides thereof and aspartate inducedsignificant amounts of CphE_(al). Subsequently, optimum concentrationsand optimum CphE_(al) harvesting time were investigated in culturesinduced with these three inductors; for CGP, concentrations between0.001-3.0 g l⁻¹ were tested, the inductive effect increased withconcentrations up to 0.05 g l⁻¹ while higher concentrations showed thesame efficiency. Maximum CphE_(al) production was obtained after 5 hfrom induction with 0.05 g l⁻¹ CGP.

Cultures cultivated under similar conditions but induced withCGP-dipeptides within the same concentration range tested for CGP,showed similar results regarding the concentration of inductor, however,maximum CphE_(al) production was obtained after only 3 h of induction.Also aspartate proved to be a suitable inductor where maximum CphE_(al)production was obtained with 4 g l⁻¹ aspartate after 5 h of induction;however, in comparison to cultures induced with optimum CGPconcentration (0.05 g l⁻¹), CphE_(al) production after induction withaspartate represented only the third.

Optimum conditions for CGP degradation to dipeptides via crudeCphE_(al): In order to decrease the handled reaction volumes and thetime required for the third phase (CGP degradation) of the originalprocess, degradation time of higher CGP concentrations (50-100 g/l) weretested at 30° C., this showed a collinear increase in degradation timewith decreasing concentrations of crude CphE_(al) and with increasingCGP concentrations. In presence of 10 g l⁻¹ crude CphE_(al), theshortest degradation period (4 h) was monitored for the lowest testedconcentration of CGP (50 g l⁻¹) while 16 h were required to degrade thehighest tested CGP concentration (100 g l⁻¹).

Experiments for estimating the optimum pH and temperature for CGPdegradation by the crude CphE_(al) powder were conducted separately asdescribed in materials and methods section. Photometrical determinationof the remaining CGP in reaction tubes indicated that maximum CGPdegradation (45%) occurred at pH 6.5, however, pH range from 5.5 to 7.5revealed close results. On the other hand, CGP degradation increasedwith increasing temperature to reach a maximum of 90% at 50° C. Thus,experiments for the optimum relation between the concentrations of CGPand CphE_(al) were repeated under the optimum parameters (50° C., pH6.5). This reduced the required degradation time (in presence of 10 gl⁻¹ crude CphE_(al)) for CGP concentrations of 50 and 100 g l⁻¹ to 1 and4 h, respectively. Also under optimum conditions, degradation timeshowed a collinear increase with decreasing concentrations of crudeCphE_(al) and with increasing CGP concentrations. This collinearrelation can be helpful to apply optimum degradation parameters duringfuture process applications. The respective formula is calculated forCGP concentrations up to 100 g l⁻¹ and based on the concentration ofpure CphE_(al) (E), for applying crude extracts, CphE_(al) content mustbe determined (s.u.) before using the formula;

P _(Degradation time at 50° C.)=17.55−29.891E_([Desired concentration of pure CphEal(g/l)])

Purification of CphE_(al) from crude supernatant powder: To move furthertowards a more efficient process and to obtain better knowledge aboutthe characteristics of the CGPase from P. alcaligenes strain DIP1,several technically applicable methods were tested to purify CphE_(al)from crude powder. Precipitation and purification of the enzyme usingsolvents such as ethanol, methanol or acetone could not providesatisfactory results due to low recovery rates and low purificationeffect. However, ammonium sulfate precipitation provided comparablybetter results with a saturation concentration of 70% but the puritygrade was rather too low.

Purification of CphE_(al) via specific substrate binding: A simplemethod was developed to purify the CGPase from P. alcaligenes strainDIP1. For this, the insoluble CGP itself was used as a binding matrix tospecifically bind CphE_(al) as described in materials and methodssection. Pre-tests showed that CphE_(al) binds completely to CGP afterthe first 5 min, therefore, this was considered as a minimum bindingtime in further applications. SDS-PAGE of all purification steps showeda gradual purification of the enzyme (FIG. 10A) and that the first twowashing steps are necessary to remove other proteins which did not bindto CGP. After degradation of CGP matrix and removing dipeptides byultrafiltration, a high purity grade of CphE_(al) was obtained.CGP-Overlay agar plates showed high activity of the purified CphE_(al)and the purified protein showed an apparent molecular weight of 45 kDain SDS-PAGE. Identity of CphE_(al) was confirmed by in-gel-renaturationwhere it regained activity and formed degradation halos on CGP-overlayagar plates. Concentration of the purified CphE_(al) was 43.2 μg ml⁻¹while the total protein content in the initial crude protein solutionwas 944 μg ml⁻¹. To detect the purity grade of the purified enzyme moreaccurately, SDS-PAGE was repeated for a larger sample volume (triplevolume) and stained for longer time with silver staining; this revealedthe presence of few other protein bands in comparably much lowerconcentrations (FIG. 10B).

Determination of CphE_(al) content in fermentation supernatants: If theapplication of pure CphE_(al) is desired in future process applications,concentration of the CGPase has to be estimated in the producedsupernatants at first, therefore, a fast test was developed using 1 mlof CGP-suspension with fixed concentration (100 mg l⁻¹) which has anOD_(600 nm) of 0.215. To this solution, 3 μl of solutions with differentconcentrations (2.7-43.2 μg ml⁻¹) of the purified enzyme were added.Reactions were incubated for 60 min at 30° C. under slow rotation (3rpm). Finally, the extent of CGP degradation was photometricallydetermined at 600 nm. To guarantee for the reproducibility of the testand the accuracy of the formula thereof (s.u.), test parameters and themeasurement range at OD₆₀₀ (0.15-0.215) must be preserved.

E _([CphEal content in crude extract(μg/ml)])=[0.2404−D_((OD600 nm after 60 min))]/0.0017

Required CGP amounts for the technical purification of CphE_(al): Tointegrate the enzyme purification procedure in the main productionprocess, also the amount of CGP, which is necessary to bind allCphE_(al) (in supernatants with known CphE_(al) content) has to bedetermined. In a total reaction volume of 0.5 ml, 50 μl of purifiedCphE_(al) solutions with different concentrations (1.4-19 μg ml⁻¹) wereadded to different CGP concentrations (0.1-6 g l⁻¹). Reaction mixtureswere incubated for 10 min at 30° C. under slow rotation (3 rpm). Aftercentrifugation (16000×g, 2 min), reaction tubes were centrifuged and thesupernatants were screened for activity on CGP-overlay agar plates. Forall tested CphE_(al) concentrations, the minimum CGP-concentrations thatdid not induce degradation halos were multiplied 3 times for safety andthen integrated in a calibration curve. The resulting formula was asfollows:

C _([required CGP concentration(g/l)]) =[E_([CphEal content in crude extract(μg/ml)])−2.4392]/0.9432

Biochemical characterization of the purified CphE_(al): The purifiedCphE_(al) showed maximum CGP degradation activity at 50° C. whilecomplete inactivation of the enzyme occurred at 68° C. Optimum pH rangefor CGP degradation was 7-8.5 with an optimum at 8.5. Testing substratespecificity of the purified CphE_(al) on CGP, BSA, bovine casein, andpoly(aspartic acid) showed the highest degradation activity on CGP.However, also BSA was partially degraded (65% in comparison to CGP).Bovine casein and poly(aspartic acid) were minimally affected by theenzymatic activity of the purified CphE_(al).

To gain information on the catalytic mechanism of CphE_(al), inhibitorexperiments were conducted employing different group-specific inhibitors(Table 1). Inhibition of the CGPase was assayed on CGP-overlay agarplates and by HPLC analysis of CGP degradation products. This revealedthat the extracellular CphE_(al) is strongly inhibited by serineprotease inhibitors Pefabloc and phenylmethylsulfonyl fluoride (PMSF).Also N-bromosuccinimide (tryptophan oxidant) led to a total inhibitionof the enzyme. In contrast, formation of degradation halos by CphE_(al)was not affected by the thiol protease inhibitor leupeptin or by themetalloprotease inhibitor EDTA. HPLC analysis confirmed these resultsexcept for samples treated with EDTA, which showed inhibition ofCphE_(al).

Example 4. Absorption of CGP dipeptides by Caco2-cell cultures: To provethe potential applications of CGP dipeptides in nutrition and therapy,the absorption of these highly soluble dipeptides was tested using cellcultures of Caco2 cells (human-colon epithelial adenocarcinoma cells) ina preliminary study. A Dulbecco's Modified Eagle Medium (DMEM)containing 20% Fetal Calf Serum (FCS) and 1% of 2 mM Glutamine solutionwas used as cultivation medium. About 7000 Caco2 cells per well weredisseminated in a 24-well plate and incubated at 37° C. under anatmosphere of 5% CO₂ and about 98% humidity. The cells were checkedmicroscopically on a daily basis until forming monolayer after nearly 50h. The incubation medium was replaced completely with fresh 700 μl perwell of the same medium supplied with 350 μg/ml of CGP dipeptides (90%Asp-Arg: 10% Asp-Lys) or with a mixture of equivalent amounts of freeL-aspartic acids, L-arginine and L-lysine. A well line was supplied withfresh 700 μl of the incubation medium without the latter additives andwas considered as control. Samples were taken after incubation periodsof 0, 13, 22, 67 and 113 h and were analyzed by HPLC as described bySallam and Steinbüchel (J. App. Microbiol. DOI:10.1111/j.1365-2672.2009.04221.x) to determine the change ofconcentrations of the tested dipeptides and amino acids.

HPLC analysis of the medium samples revealed the absorption ofapproximately 60% of cyanophycin Asp-Arg dipeptides and 68% ofcyanophycin Asp-Lys dipeptides after 113 h of incubation. On the otherhand, only 41% of free arginine and 51% of free L-lysine were absorbedover the same incubation period. Exceptionally, L-aspartic acid seems tobe better absorbed in the free form by Caco2 cells and was absorbedcompletely in a shorter incubation period of about 67 h. On thecontrary, and in comparison to the absorbed amounts of free arginine andfree lysine, 31.6% more arginine and 24.4% more lysine were absorbed bythe cells in the form of CGP dipeptides. Thus, CGP dipeptides representa higher bioavailable sources for both amino acids arginine and lysine.These results are the first direct evidence on the potential applicationof CGP dipeptides in nutrition and therapy and confirm the previousresults on the degradation of CGP by the gut flora (Sallam andSteinbüchel J. App. Microbiol. DOI: 10.1111/j.1365-2672.2009.04221.x).The results are also in agreement with previous in vitro and in vivostudies which proved that oligopeptides—in general—have higherbioavailability than their constituting amino acids in the free form(Adibi, S. A., J. Clin. Invest. 50:2266-2275 (1971); Adibi, S. A.,Gastroenterology 113:332-340 (1997); Dock, D. B. et al., Biocell28:143-150 (2004)).

TABLE 1 Wide spread, phylogeny, and characteristics of CGP degradingbacteria in mammalian, avian and fish gut flora. Halo CGP CGP Florasource/ forming degrading degrading CGP Sampling Dilution coloniesisolates isolates Identified degradation site-(Country) factor % (total)(anaerobically) isolates Oxic Anoxic Genus I. mammalian flora Bovine[Red angus; Bos taurus] Rumen (Ger) 100 x 0.9 7 4 Colon (Ger) 100 x 2.19 6 Strain 15 + ± Brevibacillus Ovine: [Swiss black- brown mountain;Pecora giurassiana] Rumen (Ger) 100 x 2.3 3 3 Strain 17 + + BacillusReticulum (Ger) 50 x 1.7 4 3 Omasum (Ger) 100 x 1.6 2 1 Abomasum (Ger)20 x 1.2 1 1 Cecum (Ger) 100 x 3 3 3 Rabbit: [European wild; Oryctolaguscuniculus] Cecum (Ger) 50 x 1.9 3 2 Strain 31 + ± Streptomyces Cecum(Egy) 50 x 3.5 1 1 II. Avian flora Duck: [Muscovy; Cairina moschata]Intestine (Ger) 50 x 0.5 2 2 Cecum (Ger) 50 x 0.6 1 1 Strain 38 + ±Micromonospora Chicken: [Langshan; Gallus Gallus] Intestine (Ger) 50 x0.8 1 Turkey: [Domestic; Meleagris gallopavo] Cecum (Egy) 50 x 1.1 3 2Strain 35 + + Bacillus Pigeon: [Common; Columba livia] Feaces (Ger) 50 x1.5 5 4 Cecum (Egy) 50 x 1.0 2 1 Strain 47 + ± Brevibacillus III. Fishflora Carp; Cyprinus carpio Digestive tract (Ger) 50 x 2.5 7 5 Strain52 + + Pseudomonas Roach; Rutilus rutilus Digestive tract (Ger) 50 x 0.82 2 Tench; Tinca tinca Digestive tract (Ger) 50 x 1.2 3 3 Telapia;Telapia nilotica Digestive tract (Egy) 50 x 2 3 2 Strain 49 + + Bacillus+; complete CGP degradation. ±; partial CGP degradation.

TABLE 2 Overview on the wide spread, habitats, and phylogeny of bacteriacapable of extracellular CGP degradation, isolated previously and duringthis study. Number of Identified Most investigated Characterized SampleSample source isolates genera strain enzymes Aerobic Gram- Marine water,Baltic sea, 11 Pseudomonas, P. Anguilliseptica CphE_(pa) negativebacteria ^(a) Pond sediment, Germany, Stryptomyces strain BI. Sewagesludge Germany Aerobic Gram- Forest soil, Germany, 43 Bacillus, B.megaterium CphE_(Bm) positive bacteria ^(b) River water GermanyMicromonospora strain BAC19 Strict anaerobic Pond sediment, Germany, 1Sedimentibacter S. hongkongensis — bacteria ^(c d) Marine water, Jordan,+27 mixed strain KI Sewage sludge, North sea, consortia Mudflats, Balticsea Soil Facoltative Pond sediment Germany 1 Pseudomonas P. Alcaligenes— anaerobic bacteria ^(e) strain DIP1 Aerobic CGP and Marine water Blacksea, 242 Flavobacterium, Flavobacterium sp. CphE_(al) asparagines Riverwater Turkey Pseudomonas, degrading bacteria ^(f) Soil Germany,Comamonas, France, Photobacter, Caucasus Oligella, Ochrobacter,Sphingomonas CGP degrading Gut flora Germany, 62 Bacillus, 8 isolates; —bacteria from Egypt Previbacillus, strains 15, 17, 31, mammalian, avianMicromonospora, 35, 38, 47, 49, 52 and fish gut flora ^(g) Stryptomyces,Pseudomonas ^(a, b, c, d) Data from Obst, M. et al., J. Biol. Chem. 277:25096-25105 (2002); Obst, M. et al., Biomacromolecules 5: 153-161(2004); Obst, M. et al., Appl. Environ. Microbiol. 71: 3642-3652 (2005)and Krug 2001, respectively. ^(e) Data from Sallam, A. et al., Submittedfor publication (2008). ^(f) Data from Zine, S., Diplom thesis, Institutfür Molekulare Mikrobiologie und Biotechnologie, WestfälischeWilhelms-Universität, Münster, Germany (2004). ^(g) This study.

TABLE 3 Effect of several group-specific inhibitors on the purifiedCphE_(al) from P. alcaligenes strain DIP1. HPLC analysis (Inhibition%^(b)) Inhibitor Concentration (mM) low inhibitor high inhibitorspecificity Inhibitor (low-high) concentration concentration Thiolproteases Leupeptin 0.001-0.01  10 39 Metalloproteases EDTA 30-60 65^(c)  75^(c) Serine proteases Pefabloc ®  8-80 91 100  Serineproteases PMSF  1-10  0 57 Tryptophan residues NBS 1-5 100  100 Control^(a) — 0 ^(a)Control without inhibitor, ^(b)The percentage valuesof inhibition refer to the control, that is, activity of CGPase in theabsence of an inhibitor, ^(c)Values are not due to inhibition but due toformation of precipitates during OPA-derivatization prior to HPLCanalysis. PMSF: Phenylmethylsulfonylfluoride, Pefabloc ®:4-(2-aminoethyl) benzensulfonylfluoride•HCl, NBS: N-bromosuccinimide.

TABLE 4 Comparison between the biochemical characteristics of CphE_(al)from P. alcaligenes strain DIP1 and previously characterized CGPases.Enzyme properties Molecular Optimum CGP Bacterium size Optimum pHdegradation (Enzyme) Localization (kDa) temperature range productsMechanism Inhibitors Synechocystis Intra- 28 35° C. 7-8  β-Asp-Arg Exo-Pefabloc ®, sp. PCC 6803 cellular peptidase PMSF, (CphB) ^(a)3,4-dichloro- isocoumarin P. anguilliseptica Extra- 43 n.d. 8-8.5β-Asp-Arg Exo- Pefabloc ®, strain BI cellular peptidase PMSF, NBS(CphE_(Pa)) ^(b) B. megaterium Extra- 37 30-42° C. 8-8.5 β-Asp-Arg +n.d. Pefabloc ®, strain BAC19 cellular (β-Asp-Arg)₂ PMSF, NBS(CphE_(Bm)) ^(c) P. alcaligenes Extra- 45 50° C. 7-8.5 β-Asp-Arg n.d.Pefabloc ®, strain DIP1 cellular PMSF, NBS (CphE_(al)) ^(d) ^(a) Datafrom Richter, R. et al., Eur. J. Biochem. 263: 163-169 (1999), ^(b) Datafrom Obst, M. et al., J. Biol. Chem. 277: 25096-25105 (2002), ^(c) Datafrom Obst, M. et al., Biomacromolecules 5: 153-161 (2004), ^(d) Thisstudy. PMSF: Phenylmethylsulfonylfluoride, Pefabloc ®: 4-(2-aminoethyl)benzensulfonylfluoride•HCl, NBS: N-bromosuccinimide, n.d.: notdetermined.

1. A process for the enzymatic production of a dipeptide compositionfrom a preparation of a cyanophycin (CGP) or CGP-like polymer beingpeptidic structures essentially comprised of one or more dipeptideunits, which process comprises degrading the polymer preparation with anCGPase from P. alcaligenes having a molecular weight of about 45 kDa, anactivity temperature range from 10 to 68° C., and an activity pH rangeof 5 to 9, or a mutant, derivative or fragment thereof capable ofcleavage of the CGP or CGP-like polymer into dipeptides.
 2. The processof claim 1, wherein the CGPase is a CGPase from P. alcaligenes strainDIP1.
 3. The process of claim 1 or 2, wherein the CGPase has an activitytemperature range from 15 to 60° C., preferably from 40-60° C., and anoptimum pH range of 7 to 8.5 and degrades CGP into its constitutingβ-dipeptides, most preferably the CGPase has a molecular weight of about45 kDa, an optimum temperature of about 50° C., and an optimum pH rangeof 7 to 8.5 and degrades CGP into β-Asp-Arg.
 4. The process of any oneof claims 1 to 3, wherein the CGPase is the P. alcaligenes DIP1 CGPaseCphE_(al) having been deposited with the DSMZ as DSM 21533, or a mutant,derivative or fragment thereof.
 5. The process of any one of claims 1 to4, wherein (i) the dipeptide units are composed of two of the followingamino acids: aspartic acid, arginine, lysine, glutamic acid, citrulline,ornithine and canevanine; and/or (ii) the dipeptide composition iscomposed of a single dipeptide or of a mixture of dipeptides; and/or(iii) the dipeptide composition is composed of dipeptides comprisingamino acid residues selected from aspartate, arginine, lysine and otheramino acid residues present in the CGP-like polymer.
 6. The process ofclaim 5, wherein the dipeptides/dipeptide units are selected fromβ-aspartate-arginine and β-aspartate-lysine.
 7. The process of any oneof claims 1 to 6, which further comprises preparing the CGP or CGP-likepolymer preparation by culturing a prokaryotic or eukaryotic producingcell line.
 8. The process of claim 7, wherein the producing cell lineselected from Escherichia coli, Ralstonia eutropha, Acinetobacterbaylyi, Corynebacterium glutamicum, Pseudomonas putida, yeast strains,and plant biomass, most preferably; Ralstonia eutropha H16-PHB⁻4-Δeda(pBBR1MCS-2::cphA₆₃₀₈/edaH16) and E. coli DH1(pMa/c5-914::cphA_(PCC6803)).
 9. The process of claim 7 or 8, whichfurther comprises isolating, purifying and/or chemically modifying theCGP product obtained by cultivating the producing cell line.
 10. Theprocess of any one of claim 7 or 8, wherein the CGP product obtained bycultivating the producing cell line is directly, without isolation orpurification subjected to degradation with the CGPase.
 11. The processof any one of claims 1 to 10, which further comprises purifying orseparating the degradation product.
 12. The process of any one of claims1 to 11, which further comprises chemically modifying the degradationproduct.
 13. A CGPase as defined in any one of claims 1 to
 4. 14. Acomposition, pharmaceutical composition, food or feed supplementcomprising dipeptides or a dipeptide mixture obtained from a cyanophycin(CGP) or a CGP-like polymer being peptidic structures essentiallycomprised of one or more dipeptide units, by enzymatic proteolysis. 15.The composition, pharmaceutical composition, medicament, food or feedsupplement of claim 14, wherein the dipeptides or dipeptide mixture isobtained from the CGP or CGP-like polymer by a process as defined inclaims 1 to
 12. 16. The composition, pharmaceutical composition,medicament, food or feed supplement of claim 14 or 15, wherein thedipeptide units are composed of two of the following amino acids:aspartic acid, arginine, lysine, glutamic acid, citrulline, ornithineand canevanine, preferably the composition, pharmaceutical composition,medicament, food or feed supplement comprises β-aspartate-arginineand/or β-aspartate-lysine.
 17. Use of a dipeptides or a dipeptidemixture as defined in claims 14 to 16 for preparing a medicament fornutritional therapy, or as a food or feed supplement.
 18. A method fornutritional therapy of a patient in need thereof, said method comprisingadministering to the patient a suitable amount of the dipeptides or thedipeptide mixture as defined in claims 14 to 16.